TWI825029B - Hla class ii-restricted t cell receptors against mutated ras - Google Patents

Abstract

Disclosed is an isolated or purified T cell receptor (TCR), wherein the TCR has antigenic specificity for mutated Kirsten rat sarcoma viral oncogene homolog (KRAS) presented by a human leukocyte antigen (HLA) Class II molecule. Related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions are also provided. Also disclosed are methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal.

Description

HLA class II-restricted T cell receptors targeting mutated RAS

Some cancers may have very limited treatment options, especially when the cancer becomes metastatic and unresectable. Despite advances in treatments such as surgery, chemotherapy, and radiation therapy, the prognosis for many cancers (such as pancreatic, colorectal, lung, endometrial, ovarian, and prostate cancers) can be poor. Therefore, there is an unmet need for other cancer treatments.

One embodiment of the invention provides an isolated or purified T cell receptor (TCR), wherein the TCR has antigenic specificity for a mutated human Ras amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutated human The RAS amino acid sequence is the mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence, the mutant human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence, or the mutant human neuroblastoma Rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

Another embodiment of the invention provides an isolated or purified polypeptide comprising a functional part of the TCR of the invention, wherein the functional part includes the following amino acid sequence: (a) all of SEQ ID NO: 1-3, (b) SEQ ID NO: all of 4-6, (c) all of SEQ ID NO: 7-9, (d) all of SEQ ID NO: 10-12, (e) all of SEQ ID NO: 1-6, or (f ) SEQ ID NO: All of 7-12.

Yet another embodiment of the invention provides an isolated or purified protein comprising at least one polypeptide of the invention.

The embodiments of the present invention further provide nucleic acids, recombinant expression vectors, host cells, cell populations and pharmaceutical compositions related to the TCR, polypeptides and proteins of the present invention.

Embodiments of the invention further provide methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal.

Cross-references to related applications

This application claims the rights and interests of U.S. Provisional Patent Application No. 62/560,930, filed on September 20, 2017, the entire content of which is incorporated herein by reference. Statement Regarding Federally Sponsored Research or Development

This invention was made with government support from the National Cancer Institute of the National Institutes of Health under Project No. BC010984. The government has certain rights in the invention. Incorporation of materials submitted electronically by reference

This article incorporates by reference the entire contents of the Computer Readable Nucleotide/Amino Acid Sequence Listing, which was submitted concurrently with this article and identified as follows: a 59,753-byte ASCII (text) file named "739664_ST25.txt", The date is September 5, 2018.

RAS family proteins belong to a large family of small GTPases. Without being bound to a particular theory or mechanism, it is believed that, when mutated, RAS proteins may be involved in signaling early in tumorigenesis in many human cancers. Single amino acid substitutions activate proteins. Mutated RAS protein products are constitutively active. Mutated RAS proteins can be expressed in various human cancers, such as pancreatic cancer (e.g., pancreatic carcinoma), colorectal cancer, lung cancer (e.g., lung adenocarcinoma), endometrial cancer, ovarian cancer (e.g., epithelial ovarian cancer), and prostate cancer ) among any of them. Human RAS family proteins include Kirsten rat sarcoma viral oncogene homolog (KRAS), Harvey rat sarcoma viral oncogene homolog (HRAS), and neuroblastoma rat sarcoma viral oncogene homolog (NRAS).

KRAS is also known as GTPase KRas, V-Ki-Ras2 Kirsten rat sarcoma viral oncogene or KRAS2. There are two transcript variants of KRAS: KRAS variant A and KRAS variant B. Wild-type (WT) KRAS variant A has the amino acid sequence of SEQ ID NO: 17. Wild-type (WT) KRAS variant B has the amino acid sequence of SEQ ID NO: 18. In the following, references to "KRAS" (mutated or unmutated (WT)) refer to variant A and variant B unless otherwise specified. Upon activation, mutant KRAS binds to guanosine 5'-triphosphate (GTP) and converts GTP to guanosine 5'-diphosphate (GDP).

HRAS is another member of the RAS protein family. HRAS is also known as Harvey rat sarcoma viral oncoprotein, V-Ha-Ras Harvey rat sarcoma viral oncogene homolog, or Ras family small GTP-binding protein H-Ras. WT HRAS has the amino acid sequence of SEQ ID NO: 19.

NRAS is another member of the RAS protein family. NRAS is also known as GTPase NRas, V-Ras neuroblastoma RAS viral oncogene homolog, or NRAS1. WT NRAS has the amino acid sequence of SEQ ID NO: 20.

One embodiment of the present invention provides an isolated or purified TCR having antigenic specificity for a mutant human RAS amino acid sequence (hereinafter "mutant RAS") presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutant human The RAS amino acid sequence is a mutant human KRAS, mutant human HRAS or mutant human NRAS amino acid sequence. In the following, unless otherwise specified, references to "TCR" also refer to functional parts and functional variants of TCR.

The TCR of the present invention can have antigenic specificity for any mutant human RAS protein, polypeptide or peptide amino acid sequence. In one embodiment of the present invention, the mutated human RAS amino acid sequence is a mutated human KRAS amino acid sequence, a mutated human HRAS amino acid sequence, or a mutated human NRAS amino acid sequence. The amino acid sequences of WT human KRAS, NRAS and HRAS proteins each have a length of 188-189 amino acid residues and are highly consistent with each other. For example, the amino acid sequence of WT human NRAS protein is 86.8% identical to that of WT human KRAS protein. Amino acid residues 1-86 of WT human NRAS protein and WT human KRAS protein are 100% identical. The amino acid sequence of WT human HRAS protein is 86.3% identical to that of WT human KRAS protein. Amino acid residues 1-94 of WT human HRAS protein and WT human KRAS protein are 100% identical. In the following, unless otherwise specified, references to "RAS" (mutated or unmutated (WT)) collectively refer to KRAS, HRAS and NRAS.

In one embodiment of the invention, a mutant human RAS amino acid sequence includes a WT RAS amino acid sequence substituted with glycine at position 12, where position 12 is individually defined by reference to the WT RAS protein. The WT RAS protein may be any of the WT KRAS protein (SEQ ID NO: 17 or 18), the WT HRAS protein (SEQ ID NO: 19), or the WT NRAS protein (SEQ ID NO: 20), as described above As illustrated, amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical, and amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are 100% identical. Therefore, the amino acid residue at position 12 in each of the WT KRAS, WT HRAS, and WT NRAS proteins is the same, that is, glycine.

Glycine at position 12 of the WT RAS amino acid sequence can be substituted by any amino acid residue except glycine. In one embodiment of the invention, the substitution is to use valine or cysteine to replace glycine at position 12 of the WT RAS amino acid sequence. In this regard, embodiments of the present invention provide TCRs with antigenic specificity for any WT RAS protein, polypeptide or peptide amino acid sequence having a G12V mutation or a G12C mutation.

Herein, mutations and substitutions of RAS are defined by reference to the amino acid sequence of the WT RAS protein. Therefore, herein, description is made by reference to an amino acid residue present at a specific position in the WT RAS protein, followed by the position number, followed by the amino acid residue that replaces that residue with the specific mutation or substitution discussed. Mutations and substitutions of RAS. A RAS amino acid sequence (eg, a RAS peptide) may include less than all of the amino acid residues of the full-length WT RAS protein. Therefore, position 12 is defined herein by reference to the WT full-length RAS protein (i.e., any of SEQ ID NOs: 17-20), and it is understood that the corresponding residue in the specific example of the RAS amino acid sequence Actual location may vary. Where the position is as defined by any of SEQ ID NO: 17-20, the term "G12" refers to the glycine typically present at position 12 of any of SEQ ID NO: 17-20 , and "G12V" indicates that the glycine normally present at position 12 of any of SEQ ID NOs: 17-20 is replaced by valine. For example, a specific example of a RAS amino acid sequence is, for example, TEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO: 29) (exemplary WT KRAS corresponding to adjacent amino acid residues 2 to 24 of SEQ ID NO: 17 peptide), "G12V" means that the underlined glycine in SEQ ID NO: 29 is replaced by valine, even though the actual position of the underlined glycine in SEQ ID NO: 29 is 11.

Examples of full-length RAS proteins with G12V or G12C mutations are set forth in Table 1 below. Table 1

In one embodiment of the invention, the TCR has antigenic specificity for a RAS peptide having the above-mentioned G12V mutation or G12C mutation, wherein the mutant RAS peptide has any length. In one embodiment of the invention, the mutant RAS peptide is of any length suitable for binding to any of the HLA class II molecules described herein. For example, the TCR may be antigenically specific for a RAS peptide having a G12V mutation or a G12C mutation, the RAS peptide having about 11 to about 30 amino acid residues, about 12 to about 24 amino acid residues, or From about 18 to about 20 amino acid residues in length. Mutated RAS peptides may include any contiguous amino acid residues of a mutant RAS protein that includes a G12V or G12C mutation. In one embodiment of the present invention, the TCR may have antigenic specificity for a RAS peptide having a G12V mutation or a G12C mutation, and the mutant RAS peptide has the following length: about 30 amino acid residues, about 29 amino acid residues residues, about 28 amino acid residues, about 27 amino acid residues, about 26 amino acid residues, about 25 amino acid residues, about 24 amino acid residues, about 23 amino acid residues, about 22 amino acid residues, about 21 amino acid residues, about 20 amino acid residues, about 19 amino acid residues, about 18 amino acid residues base, about 17 amino acid residues, about 16 amino acid residues, about 15 amino acid residues, about 14 amino acid residues, about 13 amino acid residues, about 12 Amino acid residues, about 11 amino acid residues, or a range of either two of the foregoing values. Examples of specific peptides each having a G12V mutation and recognized by the G12V TCR of the invention are set forth in Table 9.

In one embodiment of the invention, the TCR of the invention is able to recognize mutant RAS presented by HLA class II molecules. In this regard, TCRs can induce immune responses when binding to mutated RAS within the context of HLA class II molecules. The TCR of the present invention can recognize mutant RAS presented by HLA class II molecules and can also bind to HLA class II molecules in addition to mutant RAS.

In one embodiment of the invention, the HLA class II molecule is an HLA-DR molecule. HLA-DR molecules are heterodimers of α chain and β chain. The HLA-DR alpha chain can be encoded by the HLA-DRA gene. The HLA-DR β chain can be encoded by the HLA-DRB1 gene, HLA-DRB3 gene, HLA-DRB4 gene or HLA-DRB5 gene. The HLA-DR molecule can be any HLA-DR molecule. Examples of HLA-DR molecules may include, but are not limited to, HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA -DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15 and HLA-DR16. Preferably, the HLA-DR molecule is HLA-DR7 or HLA-DR11.

In one embodiment of the invention, the HLA class II molecule is an HLA-DRB1 molecule. The HLA-DRB1 molecule can be any HLA-DRB1 molecule. Examples of HLA-DRB1 molecules may include (but are not limited to) HLA-DRB1*01:01, HLA-DRB1*01:02, HLA-DRB1*01:03, HLA-DRB1*03:01, HLA-DRB1*04 :01, HLA-DRB1*04:02, HLA-DRB1*04:03, HLA-DRB1*04:04, HLA-DRB1*04:05, HLA-DRB1*04:07, HLA-DRB1*07:01 , HLA-DRB1*08:01 HLA-DRB1*08:03, HLA-DRB1*09:01, HLA-DRB1*10:01, HLA-DRB1*11:01, HLA-DRB1*11:03, HLA- DRB1*11:04, HLA-DRB1*12:01, HLA-DRB1*13:01, HLA-DRB1*13:02, HLA-DRB1*13:03, HLA-DRB1*14:01, HLA-DRB1* 15:01, HLA-DRB1*15:02 and HLA-DRB1*16:01. Preferably, the HLA class II molecule is HLA-DRB1*07:01 molecule or HLA-DRB1*11:01 molecule.

The TCRs of the present invention may provide any or more of a number of advantages, including use in receptive cell transfer when expressed by cells. Mutated RAS is expressed by cancer cells and not by normal, noncancerous cells. Without being limited to a particular theory or mechanism, it is believed that the TCRs of the present invention are designed to advantageously destroy cancer cells while minimizing or eliminating damage to normal, non-cancerous cells, thereby reducing toxicity (eg, minimizing or eliminating). Furthermore, the TCRs of the present invention may advantageously and successfully treat or prevent mutant RAS-positive cancers that are unresponsive to other types of treatments, such as chemotherapy, surgery, or radiation. For example, KRAS G12V mutations are present in approximately 27% and approximately 8% of pancreatic cancer and colorectal cancer patients, respectively, and KRAS G12C mutations are present in approximately 15% of lung cancer patients. In addition, the TCR of the present invention can recognize mutant RAS with high affinity, which can provide recognition of unmanipulated tumor cells (such as tumor cells not treated with interferon (IFN)-γ, encoded mutant RAS and HLA-DRB1*07: One or both of 01, tumor cells transfected with one or both vectors of mutated RAS and HLA-DRB1*11:01, tumor cells pulsed with a RAS peptide with G12V mutation, pulsed with G12C tumor cells with mutated RAS peptides or combinations thereof). In addition, in the United States, HLA-DRB1*07:01 and HLA-DRB1*11:01 alleles are expressed in approximately 25% and approximately 10.5% of Caucasian individuals, respectively. Therefore, the TCRs of the present invention may increase the number of immunotherapy-eligible cancer patients to include those expressing one or both of the HLA-DRB1*07:01 and HLA-DRB1*11:01 alleles and who may not be suitable for use Immunotherapy patients who recognize TCRs of RAS presented by other MHC molecules.

The phrase "antigenic specificity" as used herein means that the TCR can specifically bind with high affinity and immunologically recognize mutant RAS. For example, in conjunction with (a) pulsing with a low concentration of mutant RAS peptide (e.g., about 0.05 ng/mL to about 10 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL, 8 ng/mL , 10 ng/mL, or a range defined by either two of the foregoing values) antigen-negative, HLA class II molecule-positive target cells or (b) a nucleotide sequence encoding a mutated RAS has been introduced so that the target cells express the mutation After RAS antigen-negative and HLA class II molecule-positive target cells are co-cultured together, if about 1 × 10 ⁴ to about 1 × 10 ⁵ T cells expressing TCR secrete at least about 200 pg/mL or more (for example, 200 pg /mL or more, 300 pg/mL or more, 400 pg/mL or more, 500 pg/mL or more, 600 pg/mL or more, 700 pg/mL or more, 1000 pg/mL or more, 5,000 pg/mL or more, 7,000 pg/mL or more, 10,000 pg/mL or more, 20,000 pg/mL or more or a range defined by any two of the preceding values) IFN -γ, then the TCR can be regarded as having "antigenic specificity" for the mutant RAS. Cells expressing TCRs of the present invention may also secrete IFN-γ after co-culture with antigen-negative, HLA class II molecule-positive target cells pulsed with higher concentrations of mutant RAS peptides. The HLA class II molecule can be any HLA class II molecule described herein (eg, HLA-DRB1*07:01 molecule or HLA-DRB1*11:01 molecule).

Alternatively or additionally, in combination with (a) antigen-negative, HLA class II-positive target cells pulsed with low concentrations of mutant RAS peptide or (b) nucleotide sequences encoding mutant RAS have been introduced such that the target cells express mutant RAS. After antigen-negative and HLA class II molecule-positive target cells are co-cultured together, if the T cells expressing the TCR secrete an amount of IFN-γ that is at least twice the amount of IFN-γ expressed by the negative control, the TCR can be considered to be sensitive to the mutant RAS. "Antigenic specificity". Negative controls may be, for example, (i) T cells expressing TCR co-cultured with: (a) pulsed with the same concentration of an unrelated peptide (e.g. some other peptide with a sequence different from the mutant RAS peptide) An antigen-negative, HLA class II molecule-positive target cell, or (b) an antigen-negative, HLA class II molecule-positive target cell into which a nucleotide sequence encoding an unrelated peptide has been introduced so that the target cell expresses the irrelevant peptide; or ( ii) Untransduced T cells (e.g., derived from PBMCs that do not express TCR) cocultured with: (a) antigen-negative, HLA class II-positive target cells pulsed with the same concentration of mutant RAS peptide, or (b) an antigen-negative, HLA class II molecule-positive target cell in which a nucleotide sequence encoding a mutated RAS has been introduced such that the target cell expresses the mutated RAS. The HLA class II molecules expressed by the target cells of the negative control are the same as the HLA class II molecules expressed by the target cells co-cultured with the test T cells. The HLA class II molecule can be any HLA class II molecule described herein (eg, HLA-DRB1*07:01 molecule or HLA-DRB1*11:01 molecule). IFN-γ secretion can be measured by methods known in the art (eg, enzyme-linked immunosorbent assay, ELISA).

Alternatively, or in addition, (a) antigen-negative, HLA class II-positive target cells are pulsed with low concentrations of mutant RAS peptide or (b) nucleotide sequences encoding mutant RAS have been introduced so that the target cells express mutant RAS. After the antigen-negative and HLA class II molecule-positive target cells are co-cultured together, if compared with the number of negative control T cells secreting IFN-γ, if at least twice the number of T cells expressing TCR secrete IFN-γ, then TCR can be regarded as having "antigen specificity" for mutant RAS. HLA class II molecules, peptide concentrations, and negative controls may be as described herein for other aspects of the invention. The number of cells secreting IFN-γ can be measured by methods known in the art (eg, ELISPOT).

Alternatively or additionally, a TCR may be considered "antigen specific" for mutant RAS if T cells expressing the TCR upregulate the expression of one or more T cell activation markers, as demonstrated by, for example, flow cytometry using Measured after stimulation of target cells of RAS. Examples of T cell activation markers include 4-1BB, OX40, CD107a, CD69, and interleukins that are upregulated upon antigen stimulation (eg, tumor necrosis factor (TNF), interleukin (IL)-2, etc.).

One embodiment of the present invention provides a polypeptide chain including two polypeptides (i.e., polypeptide chains, such as the alpha (α) chain of TCR, the beta (β) chain of TCR, the gamma (γ) chain of TCR, and the delta (δ) chain of TCR). chain or combination thereof). The polypeptide of the TCR of the present invention may include any amino acid sequence, provided that the TCR has antigenic specificity for mutant RAS.

In one embodiment of the invention, the TCR includes two polypeptide chains, each of which includes a variable region containing complementarity determining regions (CDRs) 1, CDR2 and CDR3 of the TCR. In one embodiment of the invention, TCR includes a first polypeptide chain and a second polypeptide chain. The first polypeptide chain includes CDR1 (α chain CDR1) containing the amino acid sequence of SEQ ID NO: 1, CDR2 of the amino acid sequence of ID NO: 2 (CDR2 of the alpha chain) and CDR3 (CDR3 of the alpha chain) containing the amino acid sequence of SEQ ID NO: 3, and the second polypeptide chain includes the amino group of SEQ ID NO: 4 CDR1 containing the amino acid sequence (CDR1 of the β chain), CDR2 containing the amino acid sequence of SEQ ID NO: 5 (CDR2 of the β chain), and CDR3 containing the amino acid sequence of SEQ ID NO: 6 (CDR3 of the β chain).

In another embodiment of the invention, TCR includes a first polypeptide chain and a second polypeptide chain. The first polypeptide chain includes CDR1 (α chain CDR1) containing the amino acid sequence of SEQ ID NO: 7, CDR2 (α-chain CDR2) with the amino acid sequence of SEQ ID NO: 8 and CDR3 (α-chain CDR3) with the amino acid sequence of SEQ ID NO: 9, and the second polypeptide chain includes the amine containing SEQ ID NO: 10 CDR1 (beta chain CDR1) with amino acid sequence, CDR2 (beta chain CDR2) containing the amino acid sequence of SEQ ID NO: 11, and CDR3 (beta chain CDR3) containing the amino acid sequence of SEQ ID NO: 12.

In this regard, the TCR of the present invention may include any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1-12. In one embodiment of the invention, the TCR includes the following amino acid sequences: (a) all of SEQ ID NO: 1-3, (b) all of SEQ ID NO: 4-6, (c) SEQ ID NO: All of 7-9, (d) all of SEQ ID NO: 10-12, (e) all of SEQ ID NO: 1-6, or (f) all of SEQ ID NO: 7-12. In a particularly preferred embodiment, the TCR includes the following amino acid sequences: (i) all of SEQ ID NO: 1-6 or (ii) all of SEQ ID NO: 7-12.

In one embodiment of the present invention, the TCR includes the amino acid sequence of the variable region of the TCR containing the above-mentioned CDR. In this regard, the TCR may include the following amino acid sequences: SEQ ID NO: 13 (variable region of the alpha chain); SEQ ID NO: 14 (variable region of the beta chain); SEQ ID NO: 15 (variable region of the alpha chain) variable region); SEQ ID NO: 16 (variable region of the beta chain); both SEQ ID NO: 13 and 14; or both SEQ ID NO: 15 and 16. Preferably, the TCR includes the following amino acid sequences: (i) both SEQ ID NO: 13 and 14, or (ii) both SEQ ID NO: 15 and 16.

The TCR of the present invention may further include an α chain constant region and a β chain constant region. The constant regions may be derived from any suitable species (eg, human or mouse). In one embodiment of the invention, the TCR further includes murine α chain and β chain constant regions or human α chain and β chain constant regions. As used herein, the term "murine ” or “human” means that the TCR (or components thereof) is derived from mouse or human, respectively, that is, the TCR (or components thereof) is derived from or is once expressed by mouse T cells or human T cells, respectively.

One embodiment of the invention provides a chimeric TCR comprising a human variable region and a murine constant region, wherein the TCR has antigenic specificity for a mutant human RAS amino acid sequence presented by an HLA class II molecule. Murine constant regions may provide any one or more advantages. For example, murine constant regions can attenuate mispairing of the TCR of the invention with the endogenous TCR of the host cell into which the TCR of the invention is introduced. Alternatively or additionally, murine constant regions may increase the performance of the TCR of the invention compared to the same TCR with human constant regions. The chimeric TCR may include the amine of SEQ ID NO: 32 (wild-type (WT) murine alpha chain constant region), SEQ ID NO: 33 (WT murine beta chain constant region), or both SEQ ID NO: 32 and 33 amino acid sequence. Preferably, the TCR of the present invention includes the amino acid sequences of both SEQ ID NO: 32 and 33. A chimeric TCR may include any murine constant region described herein in combination with any CDR region as described herein for other aspects of the invention. In this regard, the TCR may include the following amino acid sequences: (a) all of SEQ ID NO: 1-3 and 32; (b) all of SEQ ID NO: 4-6 and 33; (c) SEQ ID NO: All of 7-9 and 32; (d) All of SEQ ID NO: 10-12 and 33; (e) All of SEQ ID NO: 1-6 and 32-33; or (f) SEQ ID NO: 7- 12 and all of 32-33. In another embodiment of the invention, a chimeric TCR may comprise any of the murine constant regions described herein in combination with any of the variable regions described herein for other aspects of the invention. In this regard, the TCR may include the following amino acid sequences: (i) both SEQ ID NO: 13 and 32; (ii) both SEQ ID NO: 14 and 33; (iii) both SEQ ID NO: 15 and 32 (iv) both SEQ ID NO: 16 and 33; (v) all of SEQ ID NO: 13-14 and 32-33; or (vi) all of SEQ ID NO: 15-16 and 32-33.

In another embodiment of the invention, the TCR includes the following amino acid sequences: SEQ ID NO: 38 (α chain with WT murine constant region), SEQ ID NO: 39 (β chain with WT murine constant region) ), SEQ ID NO: 40 (α chain with WT murine constant region), SEQ ID NO: 41 (β chain with WT murine constant region), both SEQ ID NO: 38-39, or SEQ ID NO: 40-41 both.

In one embodiment of the invention, the TCR includes an alpha chain containing a variable region and a constant region and a beta chain containing a variable region and a constant region. In this regard, the TCR may include (a) an alpha chain including the amino acid sequence of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) SEQ ID X in position 243 of NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X of position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile , Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) including SEQ ID NO: 35 The β chain of the amino acid sequence, wherein the : X at position 180 of 36 is Thr or Cys; (ii) SEQ ID NO: X at position 244 of 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) SEQ ID NO : X at position 246 of 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) SEQ ID NO: X at position 247 of 36 is Gly, Ala, Val, Leu, Ile, Pro , Phe, Met or Trp; (d) a β chain including the amino acid sequence of SEQ ID NO: 37, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; (e) (a) and (b) ); or both (f) (c) and (d).

In one embodiment of the invention, the TCR includes a substituted constant region. In this regard, a TCR may include an amino acid sequence of any of the TCRs described herein having one, two, three, or four amino acid substitutions in the constant region of one or both of the alpha and beta chains. Preferably, the TCR includes a murine constant region having one, two, three or four amino acid substitutions in the murine constant region in one or both of the alpha chain and the beta chain. In a particularly preferred embodiment, the TCR includes a murine constant region having one, two, three or four amino acid substitutions in the murine constant region of the alpha chain and one in the murine constant region of the beta chain. Amino acid substitution. In some embodiments, a TCR including a substituted constant region advantageously provides one or more of the following compared to a parental TCR including an unsubstituted (wild-type) constant region: increased recognition of mutant RAS ⁺ targets, increased host Cellular performance, reducing mispairing with endogenous TCR and increasing anti-tumor activity. Generally speaking, the substituted amino acid sequences of the murine constant regions of the TCR α chain and β chain, SEQ ID NO: 30 and 31, respectively correspond to the unsubstituted murine constant region amino acid sequences of SEQ ID NO: 32 and SEQ ID NO: 32 and 31, respectively. All or part of 33, wherein SEQ ID NO: 30 has one, two, three or four amino acid substitutions compared to SEQ ID NO: 32 and SEQ ID NO: 31 has compared to SEQ ID NO: 33 An amino acid substitution. In this regard, one embodiment of the present invention provides a TCR comprising the following amino acid sequence: (a) SEQ ID NO: 30 (constant region of α chain), wherein (i) X at position 48 is Thr or Cys; ( ii) X at position 112 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) SEQ ID NO: 31 (constant region of β chain), wherein X at position 57 is Ser or Cys; or (c) both SEQ ID NO: 30 and 31. In one embodiment of the invention, the TCR comprising SEQ ID NO: 30 does not include SEQ ID NO: 32 (unsubstituted murine constant region of the alpha chain). In one embodiment of the invention, the TCR comprising SEQ ID NO: 31 does not include SEQ ID NO: 33 (unsubstituted murine constant region of the beta chain).

In one embodiment of the invention, the substituted constant region includes a cysteine substitution in the constant region of one or both of the alpha chain and the beta chain to provide a cysteine substituted TCR. Opposite cysteines in the alpha and beta chains provide disulfide bonds that connect the constant regions of the alpha and beta chains of the substituted TCR to each other and are not present in TCRs that include unsubstituted murine constant regions. middle. In this regard, the TCR may be a cysteine-substituted TCR in which one of the native Thr at position 48 in SEQ ID NO: 32 (Thr48) and the native Ser at position 57 in SEQ ID NO: 33 (Ser57) Or both can be replaced by Cys. Preferably, both the native Thr48 of SEQ ID NO: 32 and the native Ser57 of SEQ ID NO: 33 are replaced by Cys. Examples of cysteine-substituted TCR constant region sequences are described in Table 2. In one embodiment of the invention, the TCR substituted with cysteine includes (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31 or (iii) both SEQ ID NO: 30 and 31, wherein Both SEQ ID NO: 30 and 31 are as defined in Table 2. In addition to any CDR or variable region described herein, the cysteine-substituted TCR of the invention may comprise a substituted constant region.

In one embodiment of the invention, the cysteine-substituted chimeric TCR includes a full-length alpha chain and a full-length beta chain. Examples of cysteine-substituted chimeric TCR alpha and beta chain sequences are set forth in Table 2. In one embodiment of the invention, the TCR includes (i) SEQ ID NO: 34, (ii) SEQ ID NO: 35, (iii) SEQ ID NO: 36, (iv) SEQ ID NO: 37, (v) Both SEQ ID NO: 34 and 35 or (vi) both SEQ ID NO: 36 and 37, wherein all of SEQ ID NO: 34-37 are as defined in Table 2. Table 2

In one embodiment of the invention, the substituted amino acid sequence includes one of the transmembrane (TM) domains in the constant region of one or both of the α chain and β chain of hydrophobic amino acids, Substitution of two or three amino acids, thereby providing a TCR substituted with a hydrophobic amino acid (also referred to herein as an "LVL-modified TCR"). Hydrophobic amino acid substitutions in the TM domain of a TCR can increase the hydrophobicity of the TM domain of the TCR compared to TCRs that lack hydrophobic amino acid substitutions in the TM domain. In this regard, the TCR is a TCR modified by LVL, in which one, two or three of the natural Ser112, Met114 and Gly115 of SEQ ID NO: 32 can be independently modified by Ala, Val, Leu, Ile, Pro, Phe , Met or Trp substitution; preferably Leu, Ile or Val substitution. Preferably, all three of the natural Ser112, Met114 and Gly115 of SEQ ID NO: 32 can be independently replaced by Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably by Leu, Ile Or replaced by Val. In one embodiment of the invention, the LVL-modified TCR includes (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31 or (iii) both SEQ ID NO: 30 and 31, wherein SEQ ID NO : 30 and 31 are as defined in Table 3. In addition to any CDR or variable region described herein, an LVL-modified TCR of the invention may comprise a substituted constant region.

In one embodiment of the present invention, the LVL-modified TCR includes full-length α chain and full-length β chain. Examples of LVL-modified TCR alpha and beta chain sequences are set forth in Table 3. In one embodiment of the invention, the LVL-modified TCR includes (i) SEQ ID NO: 34, (ii) SEQ ID NO: 35, (iii) SEQ ID NO: 36, (iv) SEQ ID NO: 37 , (v) both SEQ ID NO: 34 and 35 or (vi) both SEQ ID NO: 36 and 37, wherein all of SEQ ID NO: 34-37 are as defined in Table 3. table 3

In one embodiment of the invention, the substituted amino acid sequence includes a cysteine substitution in the constant region of one or both of the alpha and beta chains, and the combination includes alpha with a hydrophobic amino acid. Substitution of one, two or three amino acids in the transmembrane (TM) domain in the constant region of one or both of the chain and the beta chain (also referred to herein as "substitution with cysteine" , TCR modified by LVL"). In this regard, the TCR is a cysteine-substituted, LVL-modified chimeric TCR, in which the natural Thr48 of SEQ ID NO: 32 is replaced with Cys; one of the natural Ser112, Met114, and Gly115 of SEQ ID NO: 32 , two or three are independently substituted by Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably, they are substituted by Leu, Ile or Val; and the natural Ser57 of SEQ ID NO: 33 is substituted by Cys. Preferably, all three of the natural Ser112, Met114 and Gly115 of SEQ ID NO: 32 can be independently replaced by Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably by Leu, Ile Or replaced by Val. In one embodiment of the invention, the cysteine-substituted, LVL-modified TCR includes (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31, or (iii) SEQ ID NO: 30 and 31 Both, SEQ ID NO: 30 and 31 are as defined in Table 4. In addition to any CDR or variable region described herein, a cysteine-substituted, LVL-modified TCR of the invention may comprise a substituted constant region.

In one embodiment, the cysteine-substituted, LVL-modified TCR includes a full-length alpha chain and a full-length beta chain. In one embodiment of the invention, the TCR substituted by cysteine and modified by LVL includes (i) SEQ ID NO: 34, (ii) SEQ ID NO: 35, (iii) SEQ ID NO: 36, ( iv) SEQ ID NO: 37, (v) both SEQ ID NO: 34 and 35 or (vi) both SEQ ID NO: 36 and 37, wherein all SEQ ID NO: 34-37 are as shown in Table 4 definition. Table 4

The invention also provides polypeptides comprising a functional portion of any of the TCRs described herein. The term "polypeptide" as used herein includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds.

For polypeptides of the invention, a functional moiety may be any portion that includes and is a part of the contiguous amino acids of the TCR, provided that the functional moiety specifically binds to mutant RAS. The term "functional moiety" when used in reference to a TCR refers to any part or fragment of the TCR of the invention that retains the biological activity of the TCR from which it was derived (the parent TCR). Functional moieties encompass, for example, those portions of a TCR that retain the ability to specifically bind to mutant RAS (e.g., within the context of an HLA-DRB1*07:01 molecule or an HLA-DRB1*11:01 molecule) or to bind to a parental TCR detects, treats, or prevents cancer to a similar extent, the same extent, or to a greater extent than TCR. With reference to the parent TCR, the functional portion may include, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95%, or more of the parent TCR. .

The functional moiety may include other amino acids at the amine or carboxyl terminus of the moiety, or at both termini, that are not found in the amino acid sequence of the parent TCR. Desirably, the other amino acids do not interfere with the biological function of the functional moiety, such as specifically binding to mutant RAS and/or having the ability to detect, treat or prevent cancer, etc. More desirably, the other amino acids enhance the biological activity compared to the biological activity of the parent TCR.

The polypeptide may include a functional part of any or both of the α chain and β chain of the TCR of the present invention, for example, one of the CDR1, CDR2 and CDR3 of the variable region in the α chain and/or β chain of the TCR of the present invention. or functional parts of more. In one embodiment of the invention, the polypeptide may include SEQ ID NO: 1 (CDR1 of α chain), SEQ ID NO: 2 (CDR2 of α chain), SEQ ID NO: 3 (CDR3 of α chain), SEQ ID The amino acid sequence of NO: 4 (CDR1 of β chain), SEQ ID NO: 5 (CDR2 of β chain), SEQ ID NO: 6 (CDR3 of β chain) or a combination thereof. In another embodiment of the invention, the polypeptide may include SEQ ID NO: 7 (CDR1 of α chain), SEQ ID NO: 8 (CDR2 of α chain), SEQ ID NO: 9 (CDR3 of α chain), SEQ ID NO: The amino acid sequence of ID NO: 10 (CDR1 of β chain), SEQ ID NO: 11 (CDR2 of β chain), SEQ ID NO: 12 (CDR3 of β chain) or a combination thereof.

In this regard, the polypeptide of the present invention may include any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1-12. In one embodiment of the invention, the TCR includes the following amino acid sequences: (a) all of SEQ ID NO: 1-3, (b) all of SEQ ID NO: 4-6, (c) SEQ ID NO: All of 7-9, (d) all of SEQ ID NO: 10-12, (e) all of SEQ ID NO: 1-6, or (f) all of SEQ ID NO: 7-12. In a preferred embodiment, the polypeptide includes the following amino acid sequence: (i) all of SEQ ID NO: 1-6 or (ii) all of SEQ ID NO: 7-12.

In one embodiment of the present invention, the polypeptide of the present invention may comprise, for example, a variable region in the TCR of the present invention including a combination of the above-mentioned CDR regions. In this regard, the polypeptide may include the following amino acid sequences: (i) SEQ ID NO: 13 (variable region of the alpha chain), (ii) SEQ ID NO: 14 (variable region of the beta chain), (iii) Both SEQ ID NO: 13 and 14, (iv) SEQ ID NO: 15 (variable region of the alpha chain), (v) SEQ ID NO: 16 (variable region of the beta chain), or (vi) SEQ ID NO: Both 15 and 16. Preferably, the polypeptide includes the following amino acid sequences: (i) both SEQ ID NO: 13 and 14 or (ii) both SEQ ID NO: 15 and 16.

In one embodiment of the present invention, the polypeptide of the present invention may further comprise the above-mentioned constant region of the TCR of the present invention. In this regard, the polypeptide may further comprise the following amino acid sequences: SEQ ID NO: 32 (WT murine constant region of alpha chain), SEQ ID NO: 33 (WT murine constant region of beta chain), SEQ ID NO: 30 (substituted murine constant region of the alpha chain), SEQ ID NO: 31 (substituted murine constant region of the beta chain), both SEQ ID NOs: 32 and 33, or both SEQ ID NOs: 30 and 31. Preferably, the polypeptide further includes a combination of the amino acid sequences of SEQ ID NO: 30 and 31 or SEQ ID NO: 32 and 33 and any CDR region or variable region described herein for other aspects of the invention. In one embodiment of the invention, one or both of SEQ ID NO: 30 and 31 of the polypeptide are as defined in any one of Tables 2-4.

In one embodiment of the invention, a polypeptide of the invention may comprise the entire length of the alpha or beta chain of the TCR described herein. In this regard, the polypeptide of the invention may include the amino acid sequences of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37. Alternatively, a polypeptide of the invention may comprise both chains of the TCR described herein.

For example, the polypeptide of the present invention may include: (a) the amino acid sequence of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) SEQ ID NO : X at position 243 of 34 represents Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) SEQ ID NO: X at position 245 of 34 represents Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) the amine group of SEQ ID NO: 35 Acid sequence, wherein X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) Amino acid sequence of SEQ ID NO: 36, wherein: (i) X at position 180 of SEQ ID NO: 36 is Thr Or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 36 is Met , Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d ) The amino acid sequence of SEQ ID NO: 37, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; (e) both (a) and (b); or (f) (c) and ( d)Both. In one embodiment of the invention, any one or more of SEQ ID NO: 34-37 of the polypeptide is as defined in any one of Tables 2-4.

The invention further provides proteins comprising at least one polypeptide described herein. "Protein" means a molecule comprising one or more polypeptide chains.

In one embodiment, the protein of the present invention may comprise (a) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 1-3 and a second polypeptide chain including the amino acid sequence of SEQ ID NO: 4-6 Polypeptide chain; or (b) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 7-9 and a second polypeptide chain including the amino acid sequence of SEQ ID NO: 10-12.

In another embodiment of the invention, the protein may comprise (i) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 13 and a second polypeptide chain including the amino acid sequence of SEQ ID NO: 14 chain; or (ii) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 15 and a second polypeptide chain including the amino acid sequence of SEQ ID NO: 16.

The proteins of the invention may further comprise any of the constant regions described herein for other aspects of the invention. In this regard, in one embodiment of the present invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 32 and the second polypeptide chain may further comprise SEQ ID NO: 31 or the amino acid sequence of SEQ ID NO: 33. In one embodiment of the present invention, one or both of SEQ ID NO: 30 and 31 of the protein are as defined in any one of Tables 2-4.

Alternatively or additionally, the protein of the embodiment of the present invention may comprise (a) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr Or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met , Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b ) A second polypeptide chain including the amino acid sequence of SEQ ID NO: 35, wherein X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) A second polypeptide chain including the amino acid sequence of SEQ ID NO: 36 The first polypeptide chain, wherein: (i) X at position 180 of SEQ ID NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) position 247 of SEQ ID NO: 36 where X of 194 is Ser or Cys; (e) both (a) and (b); or (f) both (c) and (d). In one embodiment of the invention, one or more of SEQ ID NOs: 34-37 are as defined in any of Tables 2-4.

The protein of the invention may be a TCR. Or, for example, if the protein includes a single polypeptide chain containing the amino acid sequences of both SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, or if the first and/or second amino acid sequences of the protein If the polypeptide chain further includes other amino acid sequences (such as amino acid sequences encoding immunoglobulin or part thereof), the protein of the present invention can be a fusion protein. In this regard, the invention also provides fusion proteins comprising at least one polypeptide of the invention as described herein and at least one other polypeptide. The other polypeptides may be present as separate polypeptides of a fusion protein, or may be present as polypeptides expressed in-frame (tandem) with one of the polypeptides of the invention described herein. Other polypeptides may encode any peptide or proteinaceous molecule or portion thereof, including but not limited to immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules (eg, CD1a, CD1b, CD1c, CD1d), etc.

Fusion proteins may include one or more copies of a polypeptide of the invention and/or one or more copies of other polypeptides. For example, a fusion protein may include 1, 2, 3, 4, 5 or more copies of a polypeptide of the invention and/or other polypeptides. Suitable methods for preparing fusion proteins are known in the art and include, for example, recombinant methods.

In some embodiments of the invention, the TCRs, polypeptides and proteins of the invention may be represented as a single protein including a linker peptide connecting the alpha chain and the beta chain. In this regard, the TCRs, polypeptides and proteins of the invention may further include linker peptides. Linker peptides can advantageously facilitate the expression of recombinant TCRs, polypeptides and/or proteins in host cells. The linker peptide may include any suitable amino acid sequence. For example, the linker peptide may be a furin-SGSG-P2A linker including the amino acid sequence of SEQ ID NO:54. When a construct containing a linker peptide is expressed by a host cell, the linker peptide can be cleaved to produce separate alpha and beta chains. In one embodiment of the invention, a TCR, polypeptide or protein may include an amino acid sequence containing a full-length α chain, a full-length β chain, and a linker peptide positioned between the α chain and the β chain.

A protein of the invention may be a recombinant antibody or an antigen-binding portion thereof comprising at least one polypeptide of the invention as described herein. As used herein, "recombinant antibody" refers to a recombinant (eg, genetically modified) protein that includes a polypeptide of the invention and at least one of the polypeptide chains of the antibody or antigen-binding portion thereof. The polypeptide of the antibody or antigen-binding portion thereof may be a heavy chain, a light chain, a variable or constant region of a heavy chain or a light chain, a single chain variable fragment (scFv) or an Fc, Fab or F(ab) ₂ ' fragment of an antibody wait. The polypeptide chain of the antibody or antigen-binding portion thereof may exist as a separate polypeptide of the recombinant antibody. Alternatively, the polypeptide chain of the antibody or antigen-binding portion thereof may exist in the form of a polypeptide expressed in-frame (tandem) with the polypeptide of the invention. The polypeptide of the antibody or its antigen-binding portion can be a polypeptide of any antibody or any antibody fragment (including any antibody and antibody fragment described herein).

The scope of the invention includes functional variants of the TCRs, polypeptides or proteins of the invention described herein. As used herein, the term "functional variant" refers to a TCR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parent TCR, polypeptide, or protein that retains the TCR, polypeptide from which the variant was derived. or the biological activity of the protein. Functional variants encompass, for example, TCRs, polypeptides, or proteins described herein (the parent TCR, polypeptide, or protein) that retain specific binding to a mutant RAS for which the parent TCR has antigenic specificity or the parent polypeptide or protein. Those functional variants that have the ability to specifically bind) such functional variants perform such binding to a similar extent, to the same extent, or to a greater extent than the parent TCR, polypeptide, or protein. With reference to the parent TCR, polypeptide or protein, the amino acid sequence of the functional variant may be, for example, at least about 30%, about 50%, about 75%, about 80%, about 90% different from the parent TCR, polypeptide or protein, respectively. , about 95%, about 96%, about 97%, about 98%, about 99% or higher.

Functional variants may, for example, include the amino acid sequence of a parent TCR, polypeptide or protein with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with certain physical and/or chemical properties is exchanged for another amino acid with the same chemical or physical properties. For example, conservative amino acid substitutions can be one acidic amino acid replacing another acidic amino acid (such as Asp or Glu), and an amino acid with a non-polar side chain replacing another amino acid with a non-polar side chain. Acids (such as Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), basic amino acids replacing another basic amino acid (Lys, Arg, etc.), those with polar side chains The amino acid replaces another amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

Alternatively or additionally, functional variants may include the amino acid sequence of the parent TCR, polypeptide or protein having at least one non-conservative amino acid substitution. In this case, preferably, the non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, so that the biological activity of the functional variant is greater than that of the parent TCR, polypeptide or protein.

A TCR, polypeptide, or protein may consist essentially of the amino acid sequence specified or described herein, such that other components of the TCR, polypeptide, or protein (e.g., other amino acids) do not materially alter the properties of the TCR, polypeptide, or protein. biological activity. In this regard, the TCR, polypeptide or protein of the invention may, for example, consist essentially of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 34-35 or the amino acid sequences of SEQ ID NO: 36-37. Likewise, for example, a TCR, polypeptide or protein of the invention may consist essentially of (i) SEQ ID NO: 13, (ii) SEQ ID NO: 14, (iii) SEQ ID NO: 15, (iv) SEQ ID NO : 16. The amino acid sequence composition of (v) SEQ ID NO: 13 and 14 or (vi) SEQ ID NO: 15 and 16. In addition, the TCR, polypeptide or protein of the present invention may essentially consist of the following amino acid sequences: (a) any one or more of SEQ ID NO: 1-12; (b) any one or more of SEQ ID NO: 1-3 All; (c) all of SEQ ID NO: 4-6; (d) all of SEQ ID NO: 7-9; (e) all of SEQ ID NO: 10-12; (f) SEQ ID NO: 1- All of 6; or (g) all of SEQ ID NO: 7-12.

The TCR, polypeptides and proteins of the invention can be of any length and can include any number of amino acids, provided that the TCR, polypeptide or protein retains its biological activity (e.g., the ability to specifically bind to mutant RAS; detecting mammals cancer; or treatment or prevention of cancer in mammals, etc.). For example, the length of the polypeptide can range from about 50 to about 5000 amino acids, such as about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 300 in length. , about 400, about 500, about 600, about 700, about 800, about 900, about 1000 or more amino acids. In this regard, the polypeptides of the invention also include oligopeptides.

The TCRs, polypeptides and proteins of the invention may include synthetic amino acids in place of one or more natural amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, alpha-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine Amino acid, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4 -Carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline Indole-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonate, aminomalonate monoamide, N'-benzyl-N'-methyl -Lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α- Aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine and α-tert-Butylglycine.

The TCRs, polypeptides and proteins of the invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-chelated, cyclized, or converted into acid addition salts via, for example, disulfide bridges. /Or as the case may be, dimerization or polymerization or coupling occurs.

The TCR, polypeptide and/or protein of the present invention can be obtained by methods known in the industry (such as de novo synthesis). Likewise, polypeptides and proteins can be produced recombinantly using the nucleic acids described herein and using standard recombinant methods. See, for example, Molecular Cloning: A Laboratory Manual , 4th edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, the TCRs, polypeptides, and/or proteins described herein may be synthesized commercially by companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA) . In this regard, the TCRs, polypeptides and proteins of the invention may be synthetic, recombinant, isolated and/or purified.

The scope of the invention includes conjugates containing any TCR, polypeptide or protein (including any functional part or variant thereof), nucleic acid, recombinant expression vector, host cell, host cell population or antibody or antigen-binding portion thereof of the invention ( such as bioconjugates). Conjugates and general methods of synthesizing the conjugates are known in the art.

One embodiment of the invention provides a nucleic acid comprising a nucleotide sequence encoding any TCR, polypeptide or protein described herein. "Nucleic acid" as used herein includes "polynucleotide", "oligonucleotide" and "nucleic acid molecule" and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, which may contain natural, non- Natural or altered nucleotides, and which may contain natural, non-natural or altered inter-nucleotide linkages (such as phosphatide linkages or phosphorothioate linkages) instead of those found in unmodified oligonucleotides Phosphodiesters between acid nucleotides. In one embodiment, the nucleic acid includes complementary DNA (cDNA). It is generally preferred that the nucleic acid does not include any insertions, deletions, inversions and/or substitutions. However, in some cases, as discussed herein, a nucleic acid may be adapted to include one or more insertions, deletions, inversions and/or substitutions.

Preferably, the nucleic acid of the present invention is recombinant. As used herein, the term "recombinant" means: (i) a molecule constructed outside a living cell by joining natural or synthetic nucleic acid segments to a nucleic acid molecule that can replicate in a living cell, or (ii) a molecule Derived from reproduction of those parts set out in (i) above. For purposes herein, replication may be in vitro or in vivo.

Nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Green and Sambrook et al. (supra). For example, nucleic acids can be chemically synthesized using natural nucleotides or nucleotides modified in various ways that are designed to increase the biological stability of the molecule or to increase the number of pairs formed upon hybridization. Physical stability of strands (such as phosphorothioate derivatives and acridine-substituted nucleotides). Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-ethyl Cytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylq nucleoside, inosine, N ⁶ -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- Methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N ^6- substituted adenine, 7-methylguanine, 5-methylaminomethyluracil , 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl q nucleoside, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2 -Methylthio-N ⁶ -prenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, q nucleoside, 2-thiocytosine , 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate methyl ester, 3-(3- Amino-3-N-2-carboxypropyl)uracil and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the invention may be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

Nucleic acids may include any nucleotide sequence encoding any TCR, polypeptide or protein described herein. In one embodiment of the invention, the nucleic acid may include the nucleotide sequence of any one of SEQ ID NOs: 42-45 (Table 5). In one embodiment of the present invention, the nucleic acid includes the nucleotide sequences of both SEQ ID NO: 42-43 or both SEQ ID NO: 44-45. table 5

In one embodiment of the invention, the nucleic acid includes a codon-optimized nucleotide sequence encoding any TCR, polypeptide, or protein described herein. Without being bound to any particular theory or mechanism, it is believed that codon optimization of nucleotide sequences increases the translation efficiency of mRNA transcripts. Codon optimization of nucleotide sequences can involve replacing the native codon with another codon that encodes the same amino acid but can be translated by a tRNA that is more readily available within the cell, thereby Increase translation efficiency. Optimization of nucleotide sequences can also reduce secondary mRNA structures that interfere with translation, thereby increasing translation efficiency.

The invention also provides nucleic acids that include a nucleotide sequence that is complementary to a nucleotide sequence of any nucleic acid described herein or that hybridizes under stringent conditions to a nucleotide sequence of any nucleic acid described herein.

Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. "High stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any nucleic acid described herein) in an amount that is detectably greater than non-specific hybridization. High stringency conditions include those that can distinguish polynucleotides with the correct complementary sequence or polynucleotides that contain only a few scattered mismatches from random sequences that have a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. conditions. These small complementary regions are easier to melt than full-length complements of 14-17 bases or more, and high stringency hybridization can make them easier to distinguish. Relatively high stringency conditions include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70°C. These high stringency conditions tolerate minimal, if any, mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the performance of any TCR of the invention. It is generally understood that conditions can be made more stringent by adding increasing amounts of formamide.

The invention also provides a nucleic acid composition comprising at least about 70% or more (e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%) of any nucleic acid described herein. A nucleic acid with a nucleotide sequence that is about 96%, about 97%, about 98%, or about 99%) identical. In this regard, a nucleic acid may consist essentially of any of the nucleotide sequences described herein.

The nucleic acids of the invention can be incorporated into recombinant expression vectors. In this regard, the invention provides recombinant expression vectors comprising any nucleic acid of the invention. In one embodiment of the present invention, the recombinant expression vector includes nucleotide sequences encoding alpha chain, beta chain and linker peptide.

For the purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that includes a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and renders the construct The vector allows the host cell to express the mRNA, protein, polypeptide or peptide when contacted with the cell under conditions sufficient to cause the expression of the mRNA, protein, polypeptide or peptide within the cell. The carrier of the present invention is entirely non-natural. However, part of the carrier may be natural. The recombinant expression vector of the present invention can include any type of nucleotides, including (but not limited to) DNA and RNA, can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and can contain natural, non-natural or altered Nucleotides. Recombinant expression vectors may include natural, non-natural internucleotide linkages, or both types of linkages. Preferably, non-natural or altered nucleotides or inter-nucleotide linkages do not hinder transcription or replication of the vector.

The recombinant expression vector of the present invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. Vectors can be selected from the following groups: pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector (such as a retroviral vector). In a particularly preferred embodiment, the recombinant expression vector system MSGV1 vector is used.

Recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described, for example, in Green and Sambrook et al., supra. Expression vectors can be prepared as circular or linear constructs and contain functional replication systems in prokaryotic or eukaryotic host cells. Replication systems may be derived from, for example, ColEl, 2 μ plasmid, lambda, SV40, bovine papillomavirus, and the like.

Desirably, where appropriate and taking into account whether the vector is DNA or RNA based, the recombinant expression vector includes regulatory sequences (e.g., transcriptional and translational start and stop codons) that are critical to the host cell type (e.g., bacteria) into which the vector is intended to be introduced. , fungi, plants or animals) are specific.

Recombinant expression vectors may contain one or more marker genes, which allow selection of transformed or transfected host cells. Marker genes include biocide resistance (eg, resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic host cells (to provide protonutrients), and the like. Suitable marker genes for use in the expression vector of the present invention include, for example, neomycin/G418 resistance gene, hygromycin resistance gene, histamine resistance gene, tetracycline resistance gene gene and ampicillin resistance gene.

The recombinant expression vector may include a natural or non-natural promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide or protein or to a nucleotide sequence that is complementary to or hybridizing with a nucleotide sequence encoding a TCR, polypeptide or protein. Nucleotide sequence. Those skilled in the art are familiar with promoter selection, such as strong, weak, inducible, tissue-specific and development-specific. Similarly, combinations of nucleotide sequences and promoters are also familiar to those skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeats of murine stem cell viruses.

The recombinant expression vectors of the invention can be designed for transient expression, stable expression, or both. Likewise, recombinant expression vectors can be prepared for constitutive expression or for inducible expression.

Additionally, recombinant expression vectors can be prepared to contain suicide genes. As used herein, the term "suicide gene" refers to a gene that causes death of the cell expressing the suicide gene. A suicide gene may be a gene that confers sensitivity to an agent (eg, a drug) to the cell expressing the gene and causes cell death when the cell contacts or is exposed to the agent. Suicide genes are known in the industry and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and inducible caspase (caspase) 9-gene system.

Another embodiment of the invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that may contain the recombinant expression vector of the invention. The host cell may be a eukaryotic cell (eg, plant, animal, fungus, or algae), or may be a prokaryotic cell (eg, bacteria or protozoa). Host cells can be cultured cells or primary cells (ie, isolated directly from an organism such as a human). Host cells can be adherent cells or suspension cells, that is, cells that grow in suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For the purpose of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, such as a DH5α cell. For the purpose of producing recombinant TCRs, polypeptides or proteins, host cells are preferably mammalian cells. More preferably, the host cell is a human cell. Although the host cells can be of any cell type, derived from any type of tissue, and at any stage of development, the host cells are preferably peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC). More preferably, the host cell is a T cell.

For the purposes herein, a T cell may be any T cell, such as a cultured T cell (e.g., a primary T cell, or a T cell from a cultured T cell line (e.g., Jurkat, SupT1, etc.), or obtained from a mammal) of T cells). If obtained from a mammal, the T cells can be obtained from a variety of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. Preferably, the T cells are human T cells. T cells can be any type of T cells and can be at any developmental stage, including (but not limited to) CD4 ⁺ /CD8 ⁺ double-positive T cells, CD4 ⁺ helper T cells (such as Th ₁ and Th ₂ cells), CD4 ⁺ T cells, CD8 ⁺ T cells (eg, cytotoxic T cells), tumor-infiltrating lymphocytes (TILs), memory T cells (eg, central memory T cells and effector memory T cells), naive T cells, and the like.

The invention also provides cell populations comprising at least one host cell described herein. The population of cells can be a heterogeneous population that includes a host cell containing any of the recombinant expression vectors described and at least one other cell (e.g., a host cell that does not include any of the recombinant expression vectors (e.g., T cells) or cells other than T cells ( For example, B cells, macrophages, neutrophils, erythrocytes, liver cells, endothelial cells, epithelial cells, muscle cells, brain cells, etc.)). Alternatively, the population of cells may be a substantially homogeneous population, wherein the population primarily includes (eg, consists essentially of) host cells containing the recombinant expression vector. The population may also be a homogeneous cell population, wherein all of the population's cells are homologous to a single host cell that includes the recombinant expression vector, such that all of the population's cells include the recombinant expression vector. In one embodiment of the invention, the cell population includes a homogeneous population of host cells containing a recombinant expression vector as described herein.

In one embodiment of the invention, the number of cells in the population can be rapidly expanded. T cell numbers can be expanded by any of a number of methods known in the art, as described, for example, in: U.S. Patent 8,034,334; U.S. Patent 8,383,099; U.S. Patent Application Publication No. 2012/0244133; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). In one embodiment, T cell numbers are expanded by culturing T cells with OKT3 antibody, IL-2, and feeder PBMC (eg, irradiated allogeneic PBMC).

The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors and host cells (including populations thereof) of the present invention can be isolated and/or purified. The term "isolated" as used herein means removed from its natural environment. As used herein, the term "purified" means having increased purity, where "purity" is a relative term and need not be interpreted as absolute purity. For example, the purity can be at least about 50%, can be greater than about 60%, about 70%, about 80%, about 90%, about 95%, or can be about 100%.

The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors and host cells (including populations thereof) of the invention (hereinafter collectively referred to as the "TCR materials of the invention") can be formulated into compositions (eg, pharmaceutical compositions). In this regard, the present invention provides a pharmaceutical composition comprising any one of the TCRs, polypeptides, proteins, nucleic acids, expression vectors and host cells (including populations thereof) described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions of the present invention containing any of the TCR materials of the present invention may include more than one TCR material of the present invention (eg, polypeptides and nucleic acids) or two or more different TCRs. Alternatively, a pharmaceutical composition may comprise a TCR material of the invention in combination with another pharmaceutical active agent or drug, which is, for example, a chemotherapeutic agent, such as asparaginase, busulfan ), carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate , paclitaxel, rituximab, vinblastine, vincristine, etc.

Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier can be any commonly used for the particular TCR material of the invention contemplated. Methods of preparing administrable compositions are known or apparent to those skilled in the art and are set forth in more detail, for example, in Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press (2012). Preferably, the pharmaceutically acceptable carrier has no harmful side effects or toxicity under the conditions of use.

The choice of carrier depends in part on the particular TCR material of the invention and the particular method used to administer the TCR material of the invention. Accordingly, there are various suitable formulations of the pharmaceutical compositions of the present invention. Suitable formulations may include any for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or intraperitoneal administration. More than one route may be used to administer the TCR materials of the invention, and in some cases, a particular route may provide a more direct and efficient response than another route.

Preferably, the TCR material of the invention is administered by injection (eg intravenously). When the TCR material of the present invention is a host cell (or population thereof) that expresses the TCR of the present invention, the pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier, such as physiological saline (approximately 50% in water). 0.90% w/v NaCl, approximately 300 mOsm/L NaCl in water or approximately 9.0 g NaCl/liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA -LYTE A (Baxter, Deerfield, IL), About 5% dextrose or Ringer's lactate in water. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

For the purposes of the present invention, the amount or dosage of the TCR material of the invention (e.g., the number of cells when the TCR material of the invention is one or more cells) administered should be sufficient to produce an effect in an individual or animal within an appropriate time frame (e.g., treatment or prevention of reactions). For example, the dosage of the TCR material of the invention should be sufficient to bind to a cancer antigen (e.g., mutated RAS) or detect, treat, for a period of about 2 hours or more (e.g., 12 to 24 or more hours) from the time of administration or prevent cancer. In some embodiments, this period of time may be even longer. The dosage will depend on the potency of the particular TCR material of the invention and the condition of the animal (eg, human) and the weight of the animal (eg, human) to be treated.

A number of assays for determining administered dosage are known in the art. For purposes of the present invention, the starting dose to be administered to a mammal can be determined using an assay that includes the following steps: in a group of mammals each administered a different dose of T cells, before administering a given dose to the mammal After T cells expressing the TCR, polypeptide or protein of the present invention are compared, the degree of target cell lysis or IFN-γ secretion by these T cells is compared. The extent of target cell lysis or IFN-γ secretion upon administration of a certain dose can be analyzed by methods known in the art.

The dosage of the TCR materials of the invention will also depend on the presence, nature and extent of any adverse side effects that may accompany administration of the particular TCR material of the invention. Generally, the attending physician will consider a variety of factors in determining the dosage of the TCR material of the present invention to treat each individual patient, such as, for example, age, weight, general health, diet, gender, the patient to whom the present invention is intended to be administered. TCR material, route of administration, and severity of cancer treated. In embodiments in which the TCR material of the invention is a cell population, the number of cells administered per infusion may vary, for example, from about 1 × 10 ⁶ to about 1 × 10 ¹² cells or more. In certain embodiments, less than 1 × ¹⁰ cells may be administered.

Those skilled in the art will readily appreciate that the inventive TCR materials of the present invention can be modified in any number of ways to increase the therapeutic or preventive efficacy of the inventive TCR materials through the modifications. For example, TCR materials of the present invention can be coupled to chemotherapeutic agents, either directly or indirectly via bridges. The practice of coupling compounds to chemotherapeutic agents is known in the art. Those skilled in the art will recognize that sites on the TCR materials of the present invention that are not required for the function of the TCR materials of the present invention are suitable sites for attachment of bridges and/or chemotherapeutic agents, provided that the bridges and/or chemical The therapeutic agent, when attached to the TCR material of the invention, does not interfere with the function of the TCR material of the invention (i.e., the ability to bind to mutant RAS or detect, treat, or prevent cancer).

The pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells and cell populations of the present invention are expected to be used in methods of treating or preventing cancer. Without being bound by a particular theory, it is believed that the TCR of the invention specifically binds to mutant RAS such that the TCR (or a related polypeptide or protein of the invention), when expressed by a cell, is capable of mediating an immune response against target cells expressing mutant RAS. In this regard, the present invention provides a method of treating or preventing cancer in a mammal, which includes administering to the mammal: any pharmaceutical composition described herein, a TCR, Polypeptide or protein; any nucleic acid or recombinant expression vector including a nucleotide sequence encoding any TCR, polypeptide, or protein described herein; or any recombinant vector including any recombinant vector encoding any TCR, polypeptide, or protein described herein host cell or cell population.

One embodiment of the present invention provides the following substances for treating or preventing cancer in mammals: any pharmaceutical composition, TCR, polypeptide or protein described herein; any composition encoding any TCR, polypeptide, protein described herein A nucleic acid or recombinant expression vector containing a nucleotide sequence; or any host cell or cell population including a recombinant vector encoding any TCR, polypeptide or protein described herein.

The terms "treatment" and "prevention" and words derived therefrom as used herein do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that those skilled in the art would recognize as having potential benefits or therapeutic effects. In this regard, the methods of the present invention may provide any amount of any degree of cancer treatment or prevention in a mammal. Additionally, treatment or prevention provided by the methods of the present invention may comprise treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention may include promoting tumor regression. Likewise, for the purposes of this article, "prevention" may include delaying the onset of cancer or its symptoms or conditions. Alternatively or additionally, "prevention" may encompass preventing or delaying the recurrence of cancer or its symptoms or conditions.

Methods of detecting the presence of cancer in mammals are also provided. The method includes: (i) combining a sample comprising one or more cells from a mammal with any of the inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations or pharmaceuticals described herein contact with a substance, thereby forming a complex; and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

For the method of the present invention for detecting cancer in mammals, the cell sample may be a sample including whole cells, lysates thereof, or whole cell lysate fractions (eg, nuclear or cytoplasmic fractions, whole protein fractions, or nucleic acid fractions).

For the purposes of the present methods of detecting cancer, contact may occur in vitro or in vivo in the mammal. Preferably, the contact is external to the body.

Likewise, the complex can be detected by any number of means known in the art. For example, the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell or cell population of the invention described herein can be labeled with a detectable label, such as a radioactive isotope, a fluorophore (such as Fluorescent yellow isothiocyanate (FITC), phycoerythrin (PE)), enzymes (such as alkaline phosphatase, horseradish peroxidase) and elemental particles (such as gold particles).

For the purposes of administering the methods of the invention to a host cell or population of cells, the cells may be allogeneic or autologous to the mammalian species. Preferably, the cells are mammalian autologous cells.

For the purposes of the present methods, the cancer may be any cancer, including any of the following cancers: acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal cancer, Tube cancer or anorectal cancer, eye cancer, intrahepatic cholangiocarcinoma, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytes leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, glioma, Hodgkin lymphoma, hypopharynx Cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, Penile cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer and bladder cancer. Preferred cancers are pancreatic, colorectal, lung, endometrial, ovarian or prostate cancer. Preferably, the lung cancer is lung adenocarcinoma, the ovarian cancer is epithelial ovarian cancer, and the pancreatic cancer is pancreatic adenocarcinoma. In one embodiment of the invention, the cancer expresses a mutated human RAS amino acid sequence, wherein the mutated human RAS amino acid sequence is a mutated human KRAS, mutated human HRAS or mutated human NRAS amino acid sequence. Mutated human KRAS, mutated human HRAS, and mutated human NRAS manifested by cancer may be as described herein for other aspects of the invention.

The mammal mentioned in the method of the present invention can be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to mammals of the order Rodentia (such as mice and hamsters) and mammals of the order Logomorpha (such as rabbits). ). Preferably, the mammals are from the order Carnivora, including cats (cats) and canines (dogs). More preferably, the mammal is from the order Artiodactyla (including cattle (cows) and pigs (pig)) or the order Perssodactyla (including horses (horses)). Most preferably, the mammal is of the order Primates, Ceboids or Simoids (monkeys) or of the order Anthropoid (humans and apes). Particularly preferred mammals are humans.

The following examples further illustrate the invention but of course should not be construed as limiting the scope of the invention in any way. Example 1

This example demonstrates the isolation of a TCR with antigenic specificity for human KRAS with the G12V mutation presented by the HLA-DRB1*07:01 molecule.

A TCR antigenically specific for human KRAS with the G12V mutation presented by the HLA-DRB1*07:01 molecule was isolated from a patient's endometrial tumor sample. Briefly, tumor samples were minced, digested, and frozen. Tumor lysates were thawed and left overnight in the absence of interleukins before cell sorting. T cells were sorted from tumor digests using FACS based on PD-1 and/or OX40 expression (gated on PI ^- (viable cells) > CD3+). The FACS results are shown in Figure 1. Isotype-stained cells were used as controls.

Sorted cell numbers were expanded according to the Rapid Expansion Protocol (REP) for 3.5 weeks. For REP, T cells were cultured in microtiter 96-well plates (3 cells/well) in the presence of OKT3 antibody, IL-2, and irradiated allogeneic PBMC.

Expanded numbers of cells are pooled and autologous dendrites are pulsed with pooled 25-mer peptides or peptides encoded by 25-mer tandem minigenes (TMG) covering various tumor-specific mutations detected in the patient's tumor. DCs were tested for reactivity. Each pool contains 17-21 peptides or TMGs respectively. Interferon-γ (IFN-γ) secretion was measured by enzyme-linked immunospot (ELISPOT). The results are shown in Figure 2. As shown in Figure 2, pooled effector autologous T cells in cultures 7 and 8 recognized target DC pulsed with peptide pool 1 (PP1) and peptide pool 2 (PP2).

Mutation-responsive T cell cultures were tested against autologous DC pulsed with each single peptide from the pool of relevant peptides. Figure 3 shows what was obtained after co-culturing a No. 7 autologous T cell culture (W7) with pulsed autologous DCs with each of peptides 1-17 (P1-P17) from peptide pool 1 (PP1). result. As shown in Figure 3, T cells from culture 7 displayed high specificity against peptide P17. Peptide 17 (P17) encodes the KRAS ^G12V mutation.

Total RNA was isolated from cells of autologous T cell culture No. 7 (W7). Total RNA is then rapidly amplified of the 5' complementary DNA ends (5' RACE) using TCR-α and -β chain constant primers. The TCR PCR products were then separated by standard agarose gel electrophoresis and gel extraction. Sequence the product directly. The nucleotide sequences of the TCR alpha chain and beta chain variable regions are SEQ ID NO: 42 and 43 respectively. The amino acid sequences of the TCR alpha chain and beta chain variable regions are shown in Table 6. Complementarity determining regions (CDRs) are underlined. Table 6 Example 2

This example demonstrates that the TCR isolated in Example 1 recognizes the KRAS G12V peptide antigen presented in the context of HLA-DR molecules.

The nucleic acid sequence encoding the isolated G12V-reactive TCR of Example 1 (including the nucleotide sequences of SEQ ID NO: 42 and SEQ ID NO: 43) and comprising the cysteine-substituted, LVL-modified murine constant region Selected and cloned into retroviral expression vectors. The alpha chain murine constant region includes the amino acid sequence of SEQ ID NO: 30, in which X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The β-chain constant region includes the amino acid sequence of SEQ ID NO: 31, in which X at position 57 is Cys. A linker including the amino acid sequence of SEQ ID NO: 54 is positioned between the alpha chain constant region and the beta chain variable region. Allogeneic T cells are transduced using retroviral expression vectors.

Transduced cells (effector cells) were pulsed with target autologous antigen-presenting cells (APC) KRAS ^G12V peptide (1 ng/mL) and HLA-blocking antibody W6/32 (anti-HLA-A, -B, - C), IVA12 (pan-specific, anti-HLA class II), B7/21 (anti-HLA-DP), HB55 (anti-HLA-DR) or SPV-L3 (HLA-DQ) (target cells) were co-cultured together. Effector-transduced cells cultured alone, with DMSO or phorbol myristate acetate (PMA) were used as controls. Effector cells transduced with empty vector (mock) co-cultured with target autologous APC pulsed with 1 ng/mL KRAS G12V peptide (SEQ ID NO: 53) were used as a further control.

Effector cell reactivity against target cells (gated on CD3+ mTCR β chain+ cells) was measured by 4-1BB expression using flow cytometry. The results are shown in Figure 4. As shown in Figure 4, IVA12 and HB55 antibodies blocked effector cell reactivity against target cells, thereby instructing the transduced effector cells to recognize the KRAS G12V peptide antigen presented in a background of HLA-DR molecules.

Example 3 This example demonstrates that the TCR of Example 2 recognizes the KRAS G12V peptide antigen presented in the HLA-DRB1*07:01 molecular context.

Allogeneic T cells (effector cells) transduced with the Example 2 TCR were co-administered with autologous APCs from the Example 1 patient or APCs from a donor with the DRB1 01:01 or DRB1 07:01 haplotype (target cells). Cultivate. Target cells were pulsed with KRAS ^G12V peptide (SEQ ID NO: 53) or WT KRAS peptide (SEQ ID NO: 55). Effector cells were cocultured with APCs from an HLA-DRB1 positive donor (as a control) where one but not both donor alleles were DRB1*07:01 ("DRB mismatch"). Effector cells cultured alone, with DMSO or with PMA-ionomycin were used as additional controls. IFN-γ secretion was measured by ELISPOT. Count the number of positive wells. The results are shown in Table 7 and Figure 5. In Table 7, "TNTC" stands for "Too Many to Count." The percentage of mTCR-expressing cells expressing 4-1BB was also measured by flow cytometry. The results are shown in Figure 5. The results demonstrate that the TCR is specifically reactive against mutant KRAS represented by HLA-DRB*07:01. Table 7 Example 4

This example demonstrates the isolation of a TCR with antigenic specificity for human KRAS with the G12C mutation presented by the HLA-DRB1*11:01 molecule.

Identification of KRAS ^G12C -reactive TCRs using repeated in vitro sensitization (IVS) of peripheral blood T cell subsets from ovarian cancer patients with KRAS ^G12C -expressing tumors.

Autologous DCs were pulsed with G12C mutated peptide (SEQ ID NO: 56) and co-cultured with a subset of sorted T cells for 10 days, and then tested for reactivity, as set forth in Example 1.

To further enrich reactive cells, fractions reactive to the KRAS mutant peptide were sorted based on 4-1BB/OX40 performance and restimulated with the mutant peptide. Reactive T cells were sorted and sequenced based on 4-1BB/OX40 expression.

Total RNA was isolated from cells. Total RNA is then rapidly amplified of the 5' complementary DNA ends (5' RACE) using TCR-α and -β chain constant primers. The TCR PCR products were then separated by standard agarose gel electrophoresis and gel extraction. Sequence the product directly. The nucleotide sequences of the TCR alpha chain and beta chain variable regions are SEQ ID NO: 44 and 45 respectively. The amino acid sequences of the TCR alpha chain and beta chain variable regions are shown in Table 8. Complementarity determining regions (CDRs) are underlined. Table 8

Example 5 This example demonstrates that the TCR isolated in Example 4 recognizes the KRAS G12C peptide antigen presented in the context of HLA-DR molecules.

A nucleic acid sequence encoding the isolated G12C reactive TCR of Example 4 (including the nucleotide sequences of SEQ ID NO: 44 and SEQ ID NO: 45) and comprising a cysteine-substituted, LVL-modified murine constant region Selected and cloned into retroviral expression vectors. The alpha chain murine constant region includes the amino acid sequence of SEQ ID NO: 30, in which X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The β-chain constant region includes the amino acid sequence of SEQ ID NO: 31, in which X at position 57 is Cys. A linker including the amino acid sequence of SEQ ID NO: 54 is positioned between the alpha chain constant region and the beta chain variable region. Allogeneic T cells are transduced using retroviral expression vectors.

Cells are transduced after blocking membrane MHC class II molecules using antibodies against HLA-DQ, HLA-DR or HLA-DP or pan-specific antibodies against all of HLA-DP, HLA-DR and HLA-DQ (effector cells) with target autologous or allogeneic DC matched to a single HLA-DRB15:01 or HLA-DRB11:01 allele and pulsed with KRAS ^G12C 24-mer peptide (SEQ ID NO: 56) Cultivate. Effector transduced cells cultured with phorbol myristate acetate (PMA) or WT KRAS (SEQ ID NO: 55) were used as controls.

Effector cell reactivity against target cells (gated on CD3+ mTCR β chain+ cells) was measured by 4-1BB expression using flow cytometry. The results are shown in Figure 10. As shown in Figure 10, the TCR was specifically reactive against KRAS ^G12C presented by HLA-DRB*11:01.

Example 6 This example demonstrates that PBMC transduced with the KRAS ^G12C TCR of Example 5 recognize autologous DC pulsed with KRAS ^G12C peptide.

Allogeneic T cells were genetically modified using MSGV-1-retrovirus encoding the KRAS ^G12C TCR of Example 5. Autologous DC were loaded with WT KRAS (SEQ ID NO: 55) or KRAS ^G12C peptide (SEQ ID NO: 56) and co-cultured with TCR-transduced cells for 18 hours, followed by analysis of 4-1BB upregulation using flow cytometry. The results are shown in Figure 9. As shown in Figure 9, PBMC transduced with the KRAS ^G12C TCR of Example 5 recognized autologous DC pulsed with KRAS ^G12C peptide.

Example 7 This example demonstrates that the KRAS G12V mutant protein is processed and presented by DC and recognized by the TCR of Example 2.

Allogeneic T cells transduced with G12V-DRB1*07:01 in Example 2 were cocultured overnight with autologous DC pulsed with lysates of tumor cell lines expressing one of the following KRAS G12 mutations: G12R, G12C , G12D or G12V. Transduced cells co-cultured with autologous DC pulsed with cell lysates of tumor cell lines expressing WT KRAS were used as controls. Transduced cells cultured alone or with PMA or DMSO were used as additional controls. The percentage of cells that upregulate 4-1BB and/or OX40 was measured by flow cytometry. The number of cells expressing IFNγ was measured by ELISPOT (spot/2 × 10 ⁴ cells). The results are shown in Figure 6. As shown in Figure 6, the KRAS G12V mutant protein was processed and presented by DC and recognized by the TCR of Example 2.

Example 8 This example demonstrates that cells transduced with the G12V-DRB1*07:01 TCR of Example 2 specifically recognize the KRAS ^G12V peptide.

Allogeneic T cells transduced with the G12V-DRB1*07:01 TCR of Example 2 were pulsed with various concentrations of the 24-mer peptide KRAS ^G12V (SEQ ID NO: 53) or WT KRAS (SEQ ID NO: 55) Autologous DC were co-cultured overnight. The percentage of mTCRβ+CD8+4-1BB+ cells was measured by flow cytometry. The results are shown in Figure 7. As shown in Figure 7, cells transduced with the G12V-DRB1*07:01 TCR of Example 2 specifically recognized the KRAS ^G12V peptide.

Example 9 This example demonstrates that cells transduced with the G12V-DRB1*07:01 TCR of Example 2 specifically recognize various KRAS ^G12V peptides.

Allogeneic T cells transduced with the G12V-DRB1*07:01 TCR of Example 2 were co-cultured overnight with autologous DC pulsed with various concentrations of the peptides listed in Table 9 below. IFNγ secretion was measured by ELISPOT. The results are shown in Figure 8. As shown in Figure 8, although cells transduced with the G12V-DRB1*07:01 TCR of Example 2 specifically recognized all KRAS ^G12V peptides, SEQ ID NO: 52 was the best. Table 9

All references (including publications, patent applications, and patents) cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and to the same extent as if each reference was individually and specifically indicated to be incorporated by reference. The full text is set forth in this article.

Unless otherwise indicated herein or clearly contradicted by context, the terms "a" and "an" and "the" as well as "at least one" and similar referents are used in the context of describing the invention (especially in the context of the patent claims below). Any use of the term shall be construed to cover both the singular and the plural. Unless the context indicates otherwise or the context clearly contradicts it, the term "at least one" used immediately following a listing of one or more items (e.g., "at least one of A and B") shall be construed to mean a selection from the listed items. One of the listed items (A or B) or any combination of two or more of the listed items (A and B). Unless otherwise noted, the terms "includes," "has," "includes," and "contains" shall be construed as open-ended terms (i.e., meaning "including (but not limited to)"). Unless otherwise indicated herein, numerical ranges herein are intended only as a shorthand method of individually referring to each individual value falling within such range, and each individual value is incorporated into the specification as if individually recited herein. All methods set forth herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (eg, "such as") provided herein is intended merely to better illuminate the invention and does not impose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Those skilled in the art will understand variations of the preferred embodiments after reading the above description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors intend that the invention may be practiced otherwise than as specifically set forth herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Figure 1 illustrates graphical representation of experimental data (dot plots) detected by flow cytometry on cells stained for isotype (control) or expressly stained for PD-1 and/or OX40. The values in the histogram represent the percentage of cells expressing PD-1.

Figure 2 is a graph showing the number of interferon gamma (IFNg) positive spots/well in pooled cultures of effector autologous T cells (culture numbers W1-W16) co-cultured with pulsed delivery of the indicated 25-mer peptide. (PP) or a pool of peptides encoded by 25-mer tandem minigenes (TMG) covering various tumor-specific mutations are detected after target DC. Autologous T cells cultured alone, with DMSO or OKT3 antibodies were used as controls. Boxed symbols (▼) indicate pooled cultures (7 and 8) from which TCRs were isolated.

Figure 3 is a graph showing the number of IFNγ-positive spots/2 × 10 ⁴ (2E4) cells co-cultured with autologous T cells from Culture 7 (W7) pulsed with Peptide Pool 1 (PP1) Each of peptides 1-17 (P1-P17) was detected after autologous DC. Autologous T cells cultured with dimethylsulfoxide (DMSO) or OKT3 antibodies were used as controls.

Figure 4 is a graph showing the percentage of effector T cells transduced by the TCR expressing 4-1BB in Example 2 in HLA-blocking antibodies W6/32 (anti-HLA-A, -B, -C), IVA12 (pan-specific, anti-HLA class II), B7/21 (anti-HLA-DP), HB55 (anti-HLA-DR), or SPV-L3 (HLA-DQ) (target cells) with pulsed delivery of KRAS G12V Assayed after co-culture of target autologous APCs with peptide (1 ng/mL). Effector-transduced cells cultured alone, with DMSO or phorbol myristate acetate (PMA) were used as controls. Effector cells transduced with empty vector (mock) co-cultured with target autologous APC (pulsed with 1 ng/mL KRAS G12V peptide) were used as a further control.

Figure 5 is a graph showing the following results: (i) IFN-γ number/2 × 10 ⁴ cells, measured by ELISPOT; and (ii) percentage of mTCRβ+CD8+4-1BB+ cells, measured by flow cytometry Technical measurements were performed on co-cultures of TCR-transduced T cells from Example 2 with autologous APCs (4148 MB) or from donors with DRB1 01:01 or DRB1 07:01 haplotypes and pulsed with Measured after APC of KRAS ^G12V peptide or WT KRAS peptide. Effector cells were co-cultured with APCs from an HLA-DRB1 positive donor ("DRB mismatch") as a control. Effector cells cultured alone, with DMSO or phorbol myristate acetate-ionomycin (PMA: Iono) were used as additional controls.

Figure 6 is a graph showing the following results: (i) IFN-γ number/2 × 10 ⁴ cells, measured by ELISPOT (hatched lines); and (ii) percentage of cells expressing 4-1BB and/or OX40 , measured by flow cytometry (black bars), in cells co-cultured with Example 2 TCR-transduced T cells and pulsed with cell lysates of a tumor cell line expressing one of the following KRAS G12 mutations Measured after autologous DC: G12RG, 12C, G12D or G12V. Transduced cells co-cultured with autologous DC pulsed with cell lysates of tumor cell lines expressing WT KRAS were used as controls. Transduced cells cultured alone or with PMA or DMSO were used as additional controls.

Figure 7 is a graph showing the percentage of mTCRβ+CD8+4-1BB+ cells measured by flow cytometry in overnight co-cultures of Example 2 TCR-transduced T cells with pulsed delivery of KRAS ^G12V at the indicated concentrations. Measured after autologous DC of peptide (triangles) or WT KRAS peptide (squares).

Figure 8 is a graph showing the number of IFN-γ/2 × 10 ⁴ cells by ELISPOT in co-culture of Example 2 TCR-transduced T cells with autologous DC pulsed with the peptides at the concentrations indicated in Table 9 Measured later.

Figure 9 depicts experimental data (dot plot) illustrating the percentage of cells expressing murine TCR beta chain and 4-1BB in co-cultures of cells transduced with MSGV-1-retrovirus encoding the KRAS ^G12C TCR. Assayed after loading with DMSO (control) or DC with indicated WT KRAS or KRAS ^G12C peptide at the indicated concentration. Dot plots indicate the percentage of cells: mTCRβ+/4-1BB- (upper left quadrant (Q1)); mTCRβ+/4-1BB+ (upper right quadrant (Q2)); mTCRβ-/4-1BB+ (lower right quadrant (Q3) ); mTCRβ-/4-1BB- (lower left quadrant (Q4)), and are shown as follows (percentages in brackets): DMSO: Q1 (71.0), Q2 (0.96), Q3 (0.20), Q4 (27.9). WT 10 μg/ml: Q1 (64.5), Q2 (4.27), Q3 (0.43), Q4 (30.8). WT 1 μg/ml: Q1 (70.6), Q2 (1.13), Q3 (0.20), Q4 (28.1). G12C 10 μg/ml: Q1 (13.6), Q2 (51.7), Q3 (1.61), Q4 (33.0). G12C 1 μg/ml: Q1 (19.7), Q2 (46.9), Q3 (1.67), Q4 (31.7).

Figure 10 is a graph showing the percentage of cells expressing CD3 and 4-1BB after blocking membrane MHC-II using antibodies to HLA-DQ, DR, DP or all of HLA-DQ, DR and DP. Subsequently, KRAS ^G12C TCR-transduced T cells were co-expressed with autologous or allogeneic DC matched to a single HLA-DRB15:01 or HLA-DRB11:01 allele and pulsed with the KRAS ^G12C 24-mer peptide. Determination after incubation. Transduced cells co-cultured with DC pulsed with WT KRAS peptide were used as controls. Transduced cells co-cultured with PMA/ion served as another control.

<110> U.S. Department of Health and Human Services (THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES)

<120> HLA class II-restricted T cell receptor for mutated RAS

<130> 739664

<140>TW 107133221

<141> 2018-09-20

<150> US 62/560,930

<151> 2017-09-20

<160> 56

<170> PatentIn version 3.5

<210> 1

<211> 6

<212> PRT

<213> Homo sapiens

<400> 1

<210> 2

<211> 7

<212> PRT

<213> Homo sapiens

<400> 2

<210> 3

<211> 13

<212> PRT

<213> Homo sapiens

<400> 3

<210> 4

<211> 6

<212> PRT

<213> Homo sapiens

<400> 4

<210> 5

<211> 7

<212> PRT

<213> Homo sapiens

<400> 5

Claims (47)

An isolated or purified T cell receptor (TCR), wherein the TCR has antigenic specificity for a mutated human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutated human RAS amino acid sequence The acid sequence is mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), mutated human Harvey rat sarcoma viral oncogene homolog (HRAS), or mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS). Amino acid sequence, and the TCR includes: (a) α chain complementarity determining region (CDR) 1 including the amino acid sequence of SEQ ID NO: 1, α chain CDR2 including the amino acid sequence of SEQ ID NO: 2 , α chain CDR3 including the amino acid sequence of SEQ ID NO: 3, β chain CDR1 including the amino acid sequence of SEQ ID NO: 4, β chain CDR2 including the amino acid sequence of SEQ ID NO: 5, and β-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or (b) α-chain complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 7, or the amine of SEQ ID NO: 8 The α-chain CDR2 of the amino acid sequence, the α-chain CDR3 of the amino acid sequence of SEQ ID NO: 9, the β-chain CDR1 of the amino acid sequence of SEQ ID NO: 10, the amino acid sequence of SEQ ID NO: 11 The β-chain CDR2 of the sequence, and the β-chain CDR3 of the amino acid sequence of SEQ ID NO: 12. Such as the TCR of claim 1, wherein the HLA class II molecule is an HLA-DR molecule. Such as the TCR of claim 1, wherein the HLA class II molecule is an HLA-DRB1 molecule. For example, the TCR of claim 1, wherein the HLA class II molecule is an HLA-DRB1*07:01 molecule or an HLA-DRB1*11:01 molecule. The TCR of any one of claims 1 to 4, wherein the mutant human RAS amino acid sequence includes a wild-type human KRAS, wild-type human HRAS or wild-type human NRAS amino acid sequence that replaces glycine at position 12, Position 12 is defined by reference to the wild-type human KRAS, wild-type human HRAS or wild-type human NRAS protein, respectively. Such as the TCR of claim 5, wherein the substitution is to use valine or cysteine to replace glycine at position 12. The TCR of any one of claims 1 to 4, which includes: (i) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13; (ii) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14 An amino acid sequence that is at least 95% identical to the amino acid sequence; (iii) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15; (iv) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16 An amino acid sequence that is at least 95% identical to the acid sequence; (v) an amino acid sequence that is at least 95% identical to amino acids 21-131 of SEQ ID NO: 13; (vi) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14 An amino acid sequence that is at least 95% identical to acids 17-132; (vii) an amino acid sequence that is at least 95% identical to amino acids 23-132 of SEQ ID NO: 15; (viii) an amino acid sequence that is at least 95% identical to amino acids 23-132 of SEQ ID NO: 16; Amino acid sequences with at least 95% identity between amino acids 22-137; or (ix) both (i) and (ii); both (i) and (vi); both (ii) and (v); (v) and (vi) both; Both (iii) and (iv); both (iii) and (viii); both (iv) and (vii) or both (vii) and (viii). The TCR of any one of claims 1 to 4, which includes: (i) the amino acid of SEQ ID NO: 13, (ii) the amino acid of SEQ ID NO: 14, (iii) the amino acid of SEQ ID NO: 15 Amino acid, (iv) amino acid of SEQ ID NO: 16, (v) amino acid 21-131 of SEQ ID NO: 13; (vi) amino acid 17-132 of SEQ ID NO: 14; (vii) Amino acids 23-132 of SEQ ID NO: 15; (viii) Amino acids 22-137 of SEQ ID NO: 16; or (ix) both (i) and (ii); (i) and (vi) both; (ii) and (v) both; (v) and (vi) both; (iii) and (iv) both; (iii) and (viii) both; (iv) and (vii) Both or (vii) and (viii) both. The TCR of any one of claims 1 to 4, further comprising: (a) an alpha chain constant region comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 30, wherein: ( i) X at position 48 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; ( iii) X at position 114 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) A β-chain constant region including an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31, wherein the X at position 57 of SEQ ID NO: 31 is Ser or Cys; or both (c)(a) and (b). The TCR of any one of claims 1 to 4, further comprising: (a) an alpha chain constant region including the amino acid sequence of SEQ ID NO: 30, wherein: (i) position 48 of SEQ ID NO: 30 X is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) Position 114 of SEQ ID NO: 30 X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a beta chain constant region including the amino acid sequence of SEQ ID NO: 31, wherein X at position 57 of SEQ ID NO: 31 is Ser or Cys; or (c) both (a) and (b) By. The isolated or purified TCR of any one of claims 1 to 4, which includes: (a) an alpha chain including an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 34, wherein (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) position 246 of SEQ ID NO: 34 where The X at position 180 of NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) SEQ ID X at position 246 of NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) A β-chain including an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 37, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; (e) An alpha chain comprising an amino acid sequence at least 95% identical to amino acids 21-268 of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val , Leu, Ile, Pro, Phe, Met or Trp; (f) A β-chain comprising an amino acid sequence at least 95% identical to amino acids 17-305 of SEQ ID NO: 35, wherein SEQ ID NO: 35 The X at position 180 is Thr or Cys; (ii) X at position 244 in SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 244 in SEQ ID NO: 36 X at position 246 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (h) a β chain including an amino acid sequence at least 95% identical to amino acids 22-310 of SEQ ID NO: 37, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; or (i) both (a) and (b); both (a) and (f); both (b) and (e); both (e) and (f); (c) and (d) ) both; (c) and (h) both; (d) and (g) both or (g) and (h) both. The isolated or purified TCR of any one of claims 1 to 4, which includes: (a) an alpha chain including the amino acid sequence of SEQ ID NO: 34, wherein: (i) the amino acid sequence of SEQ ID NO: 34 X at position 179 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) including SEQ The β chain of the amino acid sequence of ID NO: 35, wherein X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) includes the α chain of the amino acid sequence of SEQ ID NO: 36, wherein: i) X at position 180 of SEQ ID NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; ( iii) X at position 246 of SEQ ID NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) includes the β chain of the amino acid sequence of SEQ ID NO: 37, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; (e) includes The α chain of amino acids 21-268 of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Thr Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (f) including SEQ ID NO: 35 The β chain of amino acids 17-305 of SEQ ID NO: 35, wherein the i) X at position 180 of SEQ ID NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; ( iii) X at position 246 of SEQ ID NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (h) a beta chain including amino acids 22-310 of SEQ ID NO: 37, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; or ( i) Both (a) and (b); both (a) and (f); both (b) and (e); both (e) and (f); (c) and (d) either; both (c) and (h); both (d) and (g) or both (g) and (h). An isolated or purified polypeptide comprising a functional portion of a TCR as claimed in any one of claims 1 to 12, wherein the functional portion exhibits a mutant human RAS amino acid sequence for a human leukocyte antigen (HLA) class II molecule It has antigenic specificity, wherein the mutated human RAS amino acid sequence is caused by the mutated human Kirsten rat sarcoma virus. Oncogene homolog (KRAS), mutated human Harvey rat sarcoma viral oncogene homolog (HRAS) or mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, and such functional portions thereof Including: (a) α-chain complementarity determining region (CDR) 1 including the amino acid sequence of SEQ ID NO: 1, α-chain CDR2 including the amino acid sequence of SEQ ID NO: 2, and SEQ ID NO: 3. The α-chain CDR3 of the amino acid sequence, the β-chain CDR1 of the amino acid sequence of SEQ ID NO:4, the β-chain CDR2 of the amino acid sequence of SEQ ID NO:5, and the amine of SEQ ID NO:6 β chain CDR3 of the amino acid sequence, or (b) α chain complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 7, α chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, α-chain CDR3 including the amino acid sequence of SEQ ID NO: 9, β-chain CDR1 including the amino acid sequence of SEQ ID NO: 10, β-chain CDR2 including the amino acid sequence of SEQ ID NO: 11, and β-chain CDR3 of the amino acid sequence of SEQ ID NO: 12. The isolated or purified polypeptide of claim 13, wherein the functional part includes: (i) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13; (ii) and SEQ ID NO: 13. An amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14; (iii) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15; (iv) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16; An amino acid sequence that is at least 95% identical to amino acid sequence; (v) an amino acid sequence that is at least 95% identical to amino acids 21-131 of SEQ ID NO: 13; (vi) an amino acid sequence that is at least 95% identical to amino acids 21-131 of SEQ ID NO: 14; An amino acid sequence that is at least 95% identical to amino acids 17-132; (vii) an amino acid sequence that is at least 95% identical to amino acids 23-132 of SEQ ID NO: 15 (viii) An amino acid sequence that is at least 95% identical to amino acids 22-137 of SEQ ID NO: 16; or (ix) both (i) and (ii); both (i) and (vi) both; (ii) and (v); both (v) and (vi); both (iii) and (iv); both (iii) and (viii); (iv) and (vii) both Or both (vii) and (viii). The isolated or purified polypeptide of claim 13, wherein the functional part includes: (i) the amino acid of SEQ ID NO: 13, (ii) the amino acid of SEQ ID NO: 14, (iii) the amino acid of SEQ ID NO : Amino acid of 15, (iv) Amino acid of SEQ ID NO: 16, (v) Amino acid 21-131 of SEQ ID NO: 13; (vi) Amino acid 17- of SEQ ID NO: 14 132; (vii) amino acids 23-132 of SEQ ID NO: 15; (viii) amino acids 22-137 of SEQ ID NO: 16; or (ix) both (i) and (ii); (i) ) and (vi) both; (ii) and (v) both; (v) and (vi) both; (iii) and (iv) both; (iii) and (viii) both; (iv ) and (vii) or both (vii) and (viii). The isolated or purified polypeptide of any one of claims 13 to 15, further comprising: (a) an alpha chain constant comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 30 District, wherein: (i) X at position 48 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) position 115 of SEQ ID NO: 30 where , wherein X at position 57 of SEQ ID NO: 31 is Ser or Cys; or both (c) (a) and (b). The isolated or purified polypeptide of any one of claims 13 to 15, further comprising: (a) the amino acid sequence of SEQ ID NO: 30, wherein: (i) the amino acid sequence of position 48 of SEQ ID NO: 30 X represents Thr or Cys; (ii) X at position 112 of SEQ ID NO: 30 represents Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 30 X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp ; (b) The amino acid sequence of SEQ ID NO: 31, wherein X at position 57 of SEQ ID NO: 31 is Ser or Cys; or (c) both (a) and (b). The isolated or purified polypeptide of any one of claims 13 to 15, which includes: (a) An amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) SEQ ID NO : X at position 243 of 34 represents Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) SEQ ID NO: X at position 245 of 34 represents Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) and the amine of SEQ ID NO: 35 An amino acid sequence that is at least 95% identical to the amino acid sequence, wherein the X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 36 Acid sequence, wherein: (i) X at position 180 of SEQ ID NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe , Met or Trp; (iii) X at position 246 of SEQ ID NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) An amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 37, wherein the position of SEQ ID NO: 37 X of 194 is Ser or Cys; (e) an amino acid sequence that is at least 95% identical to amino acids 21-268 of SEQ ID NO: 34, Among them: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala , Val, Leu, Ile, Pro, Phe, Met or Trp; (f) An amino acid sequence that is at least 95% identical to amino acids 17-305 of SEQ ID NO: 35, wherein position 189 of SEQ ID NO: 35 X is Ser or Cys; (g) an amino acid sequence that is at least 95% identical to amino acids 23-269 of SEQ ID NO: 36, wherein: (i) Or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 36 is Met , Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (h ) An amino acid sequence that is at least 95% identical to amino acids 22-310 of SEQ ID NO: 37, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; or (i) (a) and (b) ) both; (a) and (f) both; (b) and (e) both; (e) and (f) both; (c) and (d) both; (c) and (h) ) both; (d) and (g) both or (g) and (h) both. The isolated or purified polypeptide of any one of claims 13 to 15, which includes: (a) the amino acid sequence of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) The amino acid sequence of SEQ ID NO: 35, wherein X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) The amino acid sequence of SEQ ID NO: 36, wherein: (i) SEQ X at position 180 of ID NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) SEQ X at position 246 of ID NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile , Pro, Phe, Met or Trp; (d) The amino acid sequence of SEQ ID NO: 37, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; (e) (a) and (b) two or (f)(c) and (d) both. An isolated or purified protein having antigenic specificity for a mutant human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat Sarcoma viral oncogene homolog (KRAS), mutated human Harvey rat sarcoma viral oncogene homolog (HRAS) or mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, and wherein the The protein includes: (a) a first polypeptide chain including the following: α chain complementarity determining region (CDR) 1 including the amino acid sequence of SEQ ID NO: 1, α chain including the amino acid sequence of SEQ ID NO: 2 chain CDR2 and the α chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a second polypeptide chain comprising the following: β chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, comprising SEQ ID NO: The β-chain CDR2 of the amino acid sequence of 5 and the β-chain CDR3 of the amino acid sequence of SEQ ID NO:6; or (b) a first polypeptide chain including the following: the amino acid of SEQ ID NO:7 The sequence of the alpha chain complementarity determining region (CDR) 1, the alpha chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8 and the alpha chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9, and the second of the following Polypeptide chain: β-chain CDR1 including the amino acid sequence of SEQ ID NO: 10, β-chain CDR2 including the amino acid sequence of SEQ ID NO: 11, and β-chain including the amino acid sequence of SEQ ID NO: 12 CDR3. The isolated or purified protein of claim 20, which includes: (i) a first polypeptide chain that includes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13, and includes a first polypeptide chain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13 The amino acid sequence of ID NO: 14 is at least 95% identical to the amino group a second polypeptide chain having an acid sequence; (ii) a first polypeptide chain comprising an amino acid sequence at least 95% identical to amino acids 21-131 of SEQ ID NO: 13, and comprising a first polypeptide chain having an amino acid sequence identical to amino acids 21-131 of SEQ ID NO: 14 a second polypeptide chain having an amino acid sequence at least 95% identical to amino acids 17-132; (iii) a first polypeptide chain including an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 13 A polypeptide chain, and a second polypeptide chain including an amino acid sequence at least 95% identical to amino acids 17-132 of SEQ ID NO: 14; (iv) including amino acid 21 of SEQ ID NO: 13 -131 A first polypeptide chain that has an amino acid sequence that is at least 95% identical, and a second polypeptide chain that includes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14; (v) A first polypeptide chain that includes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15, and includes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16 the second polypeptide chain; (vi) a first polypeptide chain comprising an amino acid sequence at least 95% identical to amino acids 23-132 of SEQ ID NO: 15, and comprising an amine identical to SEQ ID NO: 16 A second polypeptide chain having an amino acid sequence that is at least 95% identical to amino acids 22-137; (vii) a first polypeptide including an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15 chain, and a second polypeptide chain including an amino acid sequence at least 95% identical to amino acids 22-137 of SEQ ID NO: 16; or (viii) including amino acids 23-13 of SEQ ID NO: 15 132 A first polypeptide chain having an amino acid sequence that is at least 95% identical, and a second polypeptide chain including an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16. The isolated or purified protein of claim 20, which includes: (i) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 13 and a second polypeptide chain including the amino acid sequence of SEQ ID NO: 14 Polypeptide chain; (ii) a first polypeptide chain including amino acids 21-131 of SEQ ID NO: 13 and a second polypeptide chain including amino acids 17-132 of SEQ ID NO: 14; (iii) A first polypeptide chain including the amino acid sequence of SEQ ID NO: 13, and a second polypeptide chain including the amino acid sequence of amino acids 17-132 of SEQ ID NO: 14; (iv) including SEQ ID The first polypeptide chain of amino acids 21-131 of NO: 13, and the second polypeptide chain including the amino acid sequence of SEQ ID NO: 14. (v) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 15, and a second polypeptide chain including the amino acid sequence of SEQ ID NO: 16; (vi) including the amino acid sequence of SEQ ID NO: 15 a first polypeptide chain of amino acids 23-132, and a second polypeptide chain including amino acids 22-137 of SEQ ID NO: 16; (vii) a third polypeptide chain including the amino acid sequence of SEQ ID NO: 15 a polypeptide chain, and a second polypeptide chain including amino acids 22-137 of SEQ ID NO: 16; or (viii) a first polypeptide chain including amino acids 23-132 of SEQ ID NO: 15, And a second polypeptide chain including the amino acid sequence of SEQ ID NO: 16. The isolated or purified protein of any one of claims 20 to 22, further comprising: (a) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 30 Peptide chain, wherein: (i) X at position 48 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) position 115 of SEQ ID NO: 30 where Chain, wherein X at position 57 of SEQ ID NO: 31 is Ser or Cys; or both (c) (a) and (b). The isolated or purified protein of any one of claims 20 to 22, further comprising: (a) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 30, wherein: (i) SEQ ID NO. X at position 48 of NO: 30 is Thr or Cys; (ii) SEQ ID NO: X at position 112 of 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) SEQ ID X at position 114 of NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a second polypeptide chain including the amino acid sequence of SEQ ID NO: 31, wherein X at position 57 of SEQ ID NO: 31 is Ser or Cys; or (c) ( Both a) and (b). The isolated or purified protein of any one of claims 20 to 22, including: (a) A first polypeptide chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys ; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala , Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) includes A second polypeptide chain having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 35, wherein X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) includes the same amino acid sequence as SEQ ID NO: The first polypeptide chain with an amino acid sequence that is at least 95% identical to the amino acid sequence of NO: 36, wherein: (i) X at position 180 of SEQ ID NO: 36 is Thr or Cys; (ii) SEQ ID NO : X at position 244 of 36 represents Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) SEQ ID NO: X at position 246 of 36 represents Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) including SEQ ID NO: 37 A second polypeptide chain with an amino acid sequence that is at least 95% identical to the amino acid sequence, wherein 21-268 At least 95% identical amino acid sequence The first polypeptide chain listed, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO:34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) SEQ ID NO:34 The The second polypeptide chain, wherein the A polypeptide chain, wherein: (i) X at position 180 of SEQ ID NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro , Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 X is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (h) includes the second most amino acid sequence that is at least 95% identical to amino acids 22-310 of SEQ ID NO: 37 A peptide chain, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; or (i) both (a) and (b); both (a) and (f); (b) and (e) Both; both (e) and (f); both (c) and (d); both (c) and (h); both (d) and (g) or (g) and (h) both. The isolated or purified protein of any one of claims 20 to 22, which includes: (a) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 34, wherein: (i) SEQ ID NO : X at position 179 of 34 is Thr or Cys; (ii) SEQ ID NO: X at position 243 of 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) SEQ ID NO : X at position 245 of 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) SEQ ID NO: X at position 246 of 34 is Gly, Ala, Val, Leu, Ile, Pro , Phe, Met or Trp; (b) a second polypeptide chain including the amino acid sequence of SEQ ID NO: 35, wherein X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) including SEQ ID The first polypeptide chain of the amino acid sequence of NO: 36, wherein: (i) X at position 180 of SEQ ID NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser , Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) ) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) The second polypeptide chain including the amino acid sequence of SEQ ID NO: 37 , wherein chain, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly , Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (f) a second polypeptide chain including the amino acid sequence of amino acids 17-305 of SEQ ID NO: 35, wherein SEQ ID NO: X at position 189 of 35 is Ser or Cys; (g) The first polypeptide chain including the amino acid sequence of amino acids 23-269 of SEQ ID NO: 36, wherein: (i) X at position 180 is Thr or Cys; (ii) X at position 244 in SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 244 in SEQ ID NO: 36 X at position 246 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (h) a second polypeptide chain including the amino acid sequence of amino acids 22-310 of SEQ ID NO: 37, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; or ( i) Both (a) and (b); both (a) and (f); both (b) and (e); both (e) and (f); (c) and (d) either; both (c) and (h); both (d) and (g) or both (g) and (h). An isolated or purified nucleic acid comprising a TCR encoding a TCR as in any one of claims 1 to 12, a polypeptide as in any one of claims 13 to 19, or a protein as in any one of claims 20 to 26 Nucleotide sequence. A recombinant expression vector comprising the nucleic acid of claim 27. An isolated or purified host cell comprising the recombinant expression vector of claim 28. An isolated or purified cell population comprising the host cell of claim 29. A pharmaceutical composition comprising (a) a TCR according to any one of claims 1 to 12, a polypeptide according to any one of claims 13 to 19, a protein according to any one of claims 20 to 26, such as The nucleic acid of claim 27, the recombinant expression vector of claim 28, the host cell of claim 29, or the cell population of claim 30; and (b) a pharmaceutically acceptable carrier. A method for detecting the presence of cancer in a mammal, the method comprising: (a) contacting a sample including cells of the cancer with: a TCR as in any one of claims 1 to 12, a TCR as in claims 13 to 19 The polypeptide of any one of claims 20 to 26, the nucleic acid of claim 27, the recombinant expression vector of claim 28, the host cell of claim 29, the cell population of claim 30 or a pharmaceutical composition according to claim 31 whereby a complex is formed; and (b) detecting the complex, wherein detection of the complex is indicative of the presence of cancer in the mammal. The method of claim 32, wherein the cancer expresses a mutated human RAS amino acid sequence, wherein the mutated human RAS amino acid sequence is a mutated human KRAS, mutated human HRAS or mutated human NRAS amino acid sequence. The method of claim 33, wherein the mutant human RAS amino acid sequence includes a wild-type human KRAS, wild-type human HRAS or wild-type human NRAS amino acid sequence replacing glycine at position 12, wherein position 12 is represented by are respectively defined with reference to the wild-type human KRAS, wild-type human HRAS or wild-type human NRAS amino acid sequence. The method of claim 34, wherein the substitution uses valine or cysteine to replace glycine at position 12. The method of any one of claims 32 to 35, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence. The method of any one of claims 32 to 35, wherein the mutated human Ras amino acid sequence is a mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence. The method of any one of claims 32 to 35, wherein the mutated human RAS amino acid sequence Amino acid sequence of mutant human Harvey rat sarcoma viral oncogene homolog (HRAS). The method of any one of claims 32 to 35, wherein the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer or prostate cancer. A TCR as claimed in any one of claims 1 to 12, a polypeptide as claimed in any one of claims 13 to 19, a protein as claimed in any one of claims 20 to 26, a nucleic acid as claimed in claim 27, as claimed in claim 27 Use of the recombinant expression vector of claim 28, the host cell of claim 29, the cell population of claim 30, or the pharmaceutical composition of claim 31, for producing a medicament for treating or preventing cancer in mammals. The use of claim 40, wherein the cancer expresses a mutated human RAS amino acid sequence, wherein the mutated human RAS amino acid sequence is a mutated human KRAS, mutated human HRAS or mutated human NRAS amino acid sequence. The use of claim 41, wherein the mutant human RAS amino acid sequence includes a wild-type human KRAS, wild-type human HRAS or wild-type human NRAS amino acid sequence replacing glycine at position 12, wherein position 12 is represented by are respectively defined with reference to the wild-type human KRAS, wild-type human HRAS or wild-type human NRAS amino acid sequence. Such as the use of claim 42, wherein the substitution is to use valine or cysteine to replace glycine at position 12. The use of any one of claims 40 to 43, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma virus oncogene homolog (KRAS) amino acid sequence. The use of any one of claims 40 to 43, wherein the mutated human Ras amino acid sequence is a mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence. The use of any one of claims 40 to 43, wherein the mutated human RAS amino acid sequence is a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence. The use of any one of claims 40 to 43, wherein the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer or prostate cancer.